Compositions and methods for the treatment of hemoglobinopathies

ABSTRACT

The present invention is directed to genome editing systems, reagents and methods for the treatment of hemoglobinopathies.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 U.S. national phase application of theInternational Application No. PCT/IB2018/050712, filed on Feb. 5, 2018,which claims priority to, and the benefit of, U.S. Provisional patentapplication No. 62/455,464, filed on Feb. 6, 2017, the contents of eachof which are incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 30, 2018, isnamed PAT057603-WO-PCT_SL.txt and is 258,837 bytes in size.

BACKGROUND

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats)evolved in bacteria as an adaptive immune system to defend against viralattack. Upon exposure to a virus, short segments of viral DNA areintegrated into the CRISPR locus of the bacterial genome. RNA istranscribed from a portion of the CRISPR locus that includes the viralsequence. That RNA, which contains sequence complimentary to the viralgenome, mediates targeting of a Cas9 protein to the sequence in theviral genome. The Cas9 protein cleaves and thereby silences the viraltarget.

Recently, the CRISPR/Cas system has been adapted for genome editing ineukaryotic cells. The introduction of site-specific single (SSBs) ordouble strand breaks (DSBs) allows for target sequence alterationthrough, for example, non-homologous end joining (NHEJ) orhomology-directed repair (HDR).

SUMMARY OF THE INVENTION

Without being bound by theory, the invention is based in part on thediscovery that CRISPR systems, e.g., Cas9 CRISPR systems, e.g., asdescribed herein, can be used to modify cells (e.g., hematopoietic stemand progenitor cells (HSPCs)), for example, at a nondeltional HPFHregion, as described herein, to increase fetal hemoglobin (HbF)expression and/or decrease expression of beta globin (e.g., a betaglobin gene having a disease-causing mutation), for example in progeny,for example red blood cell progeny, of the modified cells, and that themodified cells (e.g., modified HSPCs) may be used to treathemoglobinopathies, e.g., sickle cell disease and beta thalassemia. Inone aspect, it has surprisingly been shown herein that introduction ofgene editing systems, e.g., CRISPR systems, e.g., as described herein,to cells (e.g., HSPCs), that target regions of the genome to which noknown HPFH mutation or deletion maps creates modified HSPCs (e.g., HSPCsthat comprise one or more indels, for example, as described herein) thatare able to efficiently engraft into an organism, persist long-term inthe engrafted organism, and differentiate, including into erythrocyteswith increased fetal hemoblobin expression. In addition, these modifiedHSPCs are capable of being cultured ex vivo, for example, in thepresence of a stem cell expander (for example as described herein) underconditions that cause them to expand and proliferate while maintainingstemness. When the gene editing systems, e.g., CRISPR systems, e.g, asdescribed herein, are introduced into HPSCs derived from sickle celldisease patients, the modified cells and their progeny (e.g., erythroidprogeny) surprisingly show not only upregulation of fetal hemoglobin,but also show a significant decrease in sickle beta-globin, and asignificant decrease in the number of sickle cells and increase thenumber of normal red blood cells, relative to unmodified cellpopulations.

Thus, in an aspect, the invention provides CRISPR systems (e.g., CasCRISPR systems, e.g., Cas9 CRISPR systems, e.g., S. pyogenes Cas9 CRISPRsystems) comprising one or more, e.g., one, gRNA molecule as describedherein. Any of the gRNA molecules described herein may be used in suchsystems, and in the methods and cells described herein.

In an aspect, the invention provides a gRNA molecule including a tracerand crRNA, wherein the crRNA includes a targeting domain that:

a) is complementary with a target sequence of a nondeletional HFPHregion (e.g., a human nondeletional HPFH region);

b) is complementary with a target sequence within the genomic nucleicacid sequence at Chr11:5,249,833 to Chr11:5,250,237, − strand, hg38;

c) is complementary with a target sequence within the genomic nucleicacid sequence at Chr11:5,254,738 to Chr11:5,255,164, − strand, hg38;

d) is complementary with a target sequence within the genomic nucleicacid sequence at Chr11:5,250,094-5,250,237, − strand, hg38;

e) is complementary with a target sequence within the genomic nucleicacid sequence at Chr11:5,255,022-5,255,164, − strand, hg38;

f) is complementary with a target sequence within the genomic nucleicacid sequence at Chr11:5,249,833-5,249,927, − strand, hg38;

g) is complementary with a target sequence within the genomic nucleicacid sequence at Chr11:5,254,738-5,254,851, − strand, hg38;

h) is complementary with a target sequence within the genomic nucleicacid sequence at Chr11:5,250,139-5,250,237, − strand, hg38; or

i) combinations thereof.

In embodiments, the targeting domain includes, e.g., consists of, anyone of SEQ ID NO: 1 to SEQ ID NO: 72. In embodiments, the targetingdomain includes, e.g., consists of, any one of SEQ ID NO: 1, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 45, SEQ ID NO: 46, SEQ IDNO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO:67. In embodiments, the targeting domain includes, e.g., consists of,any one of a) SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 28, SEQ ID NO: 34,SEQ ID NO: 48, SEQ ID NO: 51, or SEQ ID NO: 67; orb) SEQ ID NO: 1, SEQID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 54.In embodiments, the gRNA molecule includes a targeting domain whichincludes, e.g., consists of, SEQ ID NO: 8. In embodiments, the gRNAmolecule includes a targeting domain which includes, e.g., consists of,SEQ ID NO: 67. In embodiments, the gRNA molecule includes a targetingdomain which includes (e.g., consists of) a fragment of any of thesequences above.

In any of the aforementioned aspects and embodiments, the gRNA moleculemay further have regions and/or properties described herein. Inembodiments, the gRNA molecule includes a fragment of any of theaforementioned targeting domains. In embodiments, the targeting domainincludes, e.g., consists of, 17, 18, 19, or 20 consecutive nucleic acidsof any one of the recited targeting domain sequences. In embodiments,the 17, 18, 19, or 20 consecutive nucleic acids of any one of therecited targeting domain sequences are the 17, 18, 19, or 20 consecutivenucleic acids disposed at the 3′ end of the recited targeting domainsequence. In other embodiments, the 17, 18, 19, or 20 consecutivenucleic acids of any one of the recited targeting domain sequences arethe 17, 18, 19, or 20 consecutive nucleic acids disposed at the 5′ endof the recited targeting domain sequence. In other embodiments, the 17,18, 19, or 20 consecutive nucleic acids of any one of the recitedtargeting domain sequences do not include either the 5′ or 3′ nucleicacid of the recited targeting domain sequence. In embodiments, thetargeting domain consists of the recited targeting domain sequence.

In an aspect, including in any of the aforementioned aspects andembodiments, a portion of the crRNA and a portion of the tracr hybridizeto form a flagpole including SEQ ID NO: 182 or 183. In an aspect,including in any of the aforementioned aspects and embodiments, theflagpole further includes a first flagpole extension, located 3′ to thecrRNA portion of the flagpole, wherein said first flagpole extensionincludes SEQ ID NO: 184. In an aspect, including in any of theaforementioned aspects and embodiments, the flagpole further includes asecond flagpole extension located 3′ to the crRNA portion of theflagpole and, if present, the first flagpole extension, wherein saidsecond flagpole extension includes SEQ ID NO: 185.

In an aspect, including in any of the aforementioned aspects andembodiments, the tracr includes SEQ ID NO: 224 or SEQ ID NO: 225. In anaspect, including in any of the aforementioned aspects and embodiments,the tracr includes SEQ ID NO: 232, optionally further including, at the3′ end, an additional 1, 2, 3, 4, 5, 6, or 7 uracil (U) nucleotides. Inan aspect, including in any of the aforementioned aspects andembodiments, the crRNA includes, from 5′ to 3′, [targeting domain]−: a)SEQ ID NO:182; b) SEQ ID NO: 183; c) SEQ ID NO: 199; d) SEQ ID NO: 200;e) SEQ ID NO: 201; f) SEQ ID NO: 202; or g) SEQ ID NO: 226.

In an aspect, including in any of the aforementioned aspects andembodiments, the tracr includes, from 5′ to 3′: a) SEQ ID NO: 187; b)SEQ ID NO: 188; c) SEQ ID NO: 203; d) SEQ ID NO: 204; e) SEQ ID NO: 224;f) SEQ ID NO: 225; g) SEQ ID NO: 232; h) SEQ ID NO: 227; i) (SEQ ID NO:228; j) SEQ ID NO: 229; k) any of a) to j), above, further including, atthe 3′ end, at least 1, 2, 3, 4, 5, 6 or 7 uracil (U) nucleotides, e.g.,1, 2, 3, 4, 5, 6, or 7 uracil (U) nucleotides; l) any of a) to k),above, further including, at the 3′ end, at least 1, 2, 3, 4, 5, 6 or 7adenine (A) nucleotides, e.g., 1, 2, 3, 4, 5, 6, or 7 adenine (A)nucleotides; or m) any of a) to l), above, further including, at the 5′end (e.g., at the 5′ terminus), at least 1, 2, 3, 4, 5, 6 or 7 adenine(A) nucleotides, e.g., 1, 2, 3, 4, 5, 6, or 7 adenine (A) nucleotides.

In an aspect, including in any of the aforementioned aspects andembodiments, the targeting domain and the tracr are disposed on separatenucleic acid molecules. In an aspect, including in any of theaforementioned aspects and embodiments, the targeting domain and thetracr are disposed on separate nucleic acid molecules, and the nucleicacid molecule including the targeting domain includes SEQ ID NO: 201,optionally disposed immediately 3′ to the targeting domain, and thenucleic acid molecule including the tracr includes, e.g., consists of,SEQ ID NO: 224. In an aspect, including in any of the aforementionedaspects and embodiments, the crRNA portion of the flagpole includes SEQID NO: 201 or SEQ ID NO: 202. In an aspect, including in any of theaforementioned aspects and embodiments, the tracr includes SEQ ID NO:187 or 188, and optionally, if a first flagpole extension is present, afirst tracr extension, disposed 5′ to SEQ ID NO: 187 or 188, said firsttracr extension including SEQ ID NO: 189.

In an aspect, including in any of the aforementioned aspects andembodiments, the targeting domain and the tracr are disposed on a singlenucleic acid molecule, for example, wherein the tracr is disposed 3′ tothe targeting domain. In an aspect, the gRNA molecule includes a loop,disposed 3′ to the targeting domain and 5′ to the tracr. In embodiments,the loop includes SEQ ID NO: 186. In an aspect, including in any of theaforementioned aspects and embodiments, the gRNA molecule includes, from5′ to 3′, [targeting domain]−: (a) SEQ ID NO: 195; (b) SEQ ID NO: 196;(c) SEQ ID NO: 197; (d) SEQ ID NO: 198; (e) SEQ ID NO: 231; or (f) anyof (a) to (e), above, further including, at the 3′ end, 1, 2, 3, 4, 5, 6or 7 uracil (U) nucleotides.

In an aspect, including in any of the aforementioned aspects andembodiments, the targeting domain and the tracr are disposed on a singlenucleic acid molecule, and wherein said nucleic acid molecule includes,e.g., consists of, said targeting domain and SEQ ID NO: 231, optionallydisposed immediately 3′ to said targeting domain.

In an aspect, including in any of the aforementioned aspects andembodiments, one, or optionally more than one, of the nucleic acidmolecules including the gRNA molecule includes:

a) one or more, e.g., three, phosphorothioate modifications at the 3′end of said nucleic acid molecule or molecules;

b) one or more, e.g., three, phosphorothioate modifications at the 5′end of said nucleic acid molecule or molecules;

c) one or more, e.g., three, 2′-O-methyl modifications at the 3′ end ofsaid nucleic acid molecule or molecules;

d) one or more, e.g., three, 2′-O-methyl modifications at the 5′ end ofsaid nucleic acid molecule or molecules;

e) a 2′ O-methyl modification at each of the 4^(th)-to-terminal,3^(rd)-to-terminal, and 2^(nd)-to-terminal 3′ residues of said nucleicacid molecule or molecules;

f) a 2′ O-methyl modification at each of the 4^(th)-to-terminal,3^(rd)-to-terminal, and 2^(nd)-to-terminal 5′ residues of said nucleicacid molecule or molecules; or

f) any combination thereof.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 74;

(b) SEQ ID NO: 75; or

(c) SEQ ID NO: 76.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 77, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 77, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 78, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 78, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 79;

(b) SEQ ID NO: 80; or

(c) SEQ ID NO: 81.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 82, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 82, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 83, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 83, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 84;

(b) SEQ ID NO: 85; or

(c) SEQ ID NO: 86.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 87, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 87, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 88, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 88, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 89;

(b) SEQ ID NO: 90; or

(c) SEQ ID NO: 91.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 92, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 92, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 93, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 93, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 94;

(b) SEQ ID NO: 95; or

(c) SEQ ID NO: 96.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 97, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 97, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 98, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 98, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 99;

(b) SEQ ID NO: 100; or

(c) SEQ ID NO: 101.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 102, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 102, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 103, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 103, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 104;

(b) SEQ ID NO: 105; or

(c) SEQ ID NO:106.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 107, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 107, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 108, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 108, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 109;

(b) SEQ ID NO: 110; or

(c) SEQ ID NO: 111.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 112, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 112, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 113, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 113, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 114;

(b) SEQ ID NO: 115; or

(c) SEQ ID NO:116.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 117, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 117, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 118, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 118, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 119;

(b) SEQ ID NO: 120; or

(c) SEQ ID NO: 121.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 122, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 122, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 123, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 123, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 124;

(b) SEQ ID NO: 125; or

(c) SEQ ID NO:126.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 127, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 127, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 128, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 128, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 129;

(b) SEQ ID NO: 130; or

(c) SEQ ID NO: 131.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 132, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 132, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 133, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 133, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 134;

(b) SEQ ID NO: 135; or

(c) SEQ ID NO:136.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 137, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 137, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 138, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 138, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 139;

(b) SEQ ID NO: 140; or

(c) SEQ ID NO: 141.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 142, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 142, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 143, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 143, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 144;

(b) SEQ ID NO: 145; or

(c) SEQ ID NO:146.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 147, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 147, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 148, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 148, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 149;

(b) SEQ ID NO: 150; or

(c) SEQ ID NO: 151.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 152, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 152, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 153, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 153, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 154;

(b) SEQ ID NO: 155; or

(c) SEQ ID NO:156.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 157, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 157, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 158, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 158, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 159;

(b) SEQ ID NO: 160; or

(c) SEQ ID NO: 161.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 162, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 162, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 163, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 163, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 164;

(b) SEQ ID NO: 165; or

(c) SEQ ID NO:166.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 167, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 167, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 168, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 168, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 169;

(b) SEQ ID NO: 170; or

(c) SEQ ID NO: 171.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 172, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 172, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 173, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 173, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) SEQ ID NO: 174;

(b) SEQ ID NO: 175; or

(c) SEQ ID NO:176.

In an aspect, the invention provides a gRNA molecule, including, e.g.,consisting of, the sequence:

(a) a crRNA including, e.g., consisting of, SEQ ID NO: 177, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA including, e.g., consisting of, SEQ ID NO: 177, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA including, e.g., consisting of, SEQ ID NO: 178, and a tracrincluding, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA including, e.g., consisting of, SEQ ID NO: 178, and a tracrincluding, e.g., consisting of, SEQ ID NO: 73.

In an aspect, including in any of the aforementioned aspects andembodiments the invention provides a gRNA molecule, wherein:

a) when a CRISPR system (e.g., an RNP as described herein) including thegRNA molecule is introduced into a cell, an indel is formed at or nearthe target sequence complementary to the targeting domain of the gRNAmolecule; and/or

b) when a CRISPR system (e.g., an RNP as described herein) including thegRNA molecule is introduced into a cell, a deletion is created includingsequence, e.g., including substantially all the sequence, between asequence complementary to the gRNA targeting domain (e.g., at least 90%complementary to the gRNA targeting domain, e.g., fully complementary tothe gRNA targeting domain) in the HBG1 promoter region and a sequencecomplementary to the gRNA targeting domain (e.g., at least 90%complementary to the gRNA targeting domain, e.g., fully complementary tothe gRNA targeting domain) in the HBG2 promoter region. In embodiments,the indel does not include a nucleotide of a nondeletional HPFH ortranscription factor binding site.

In an aspect, including in any of the aforementioned aspects andembodiments, the invention provides a gRNA molecule, wherein when aCRISPR system (e.g., an RNP as described herein) including the gRNAmolecule is introduced into a population of cells, an indel is formed ator near the target sequence complementary to the targeting domain of thegRNA molecule in at least about 15%, e.g., at least about 17%, e.g., atleast about 20%, e.g., at least about 30%, e.g., at least about 40%,e.g., at least about 50%, e.g., at least about 55%, e.g., at least about60%, e.g., at least about 70%, e.g., at least about 75%, of the cells ofthe population. In an aspect, including in any of the aforementionedaspects and embodiments, the indel includes at least one nucleotide ofan HBG1 promoter region or at least one nucleotide of an HBG2 promoterregion. In embodiments, at least about 15% of the cells of thepopulation include an indel which includes at least one nucleotide of anHBG1 promoter region and an indel which includes at least one nucleotideof an HBG2 promoter region. In an aspect, including in any of theaforementioned aspects and embodiments, the percentage of the cells ofthe population which include an indel which includes at least onenucleotide of an HBG1 promoter region differs from percentage of thecells of the population which include an indel which includes at leastone nucleotide of an HBG2 promoter region by at least about 5%, e.g., atleast about 10%, e.g., at least about 20%, e.g., at least about 30%. Inembodiments, the indel is as measured by next generation sequencing(NGS).

In an aspect, including in any of the aforementioned aspects andembodiments, the invention provides a gRNA molecule, wherein when aCRISPR system (e.g., an RNP as described herein) including the gRNAmolecule is introduced into a cell, expression of fetal hemoglobin isincreased in said cell or its progeny, e.g., its erythroid progeny,e.g., its red blood cell progeny. In embodiments, when a CRISPR system(e.g., an RNP as described herein) including the gRNA molecule isintroduced into a population of cells, the percentage of F cells in saidpopulation or population of its progeny, e.g., its erythroid progeny,e.g., its red blood cell progeny, is increased by at least about 15%,e.g., at least about 17%, e.g., at least about 20%, e.g., at least about25%, e.g., at least about 30%, e.g., at least about 35%, e.g., at leastabout 40%, relative to the percentage of F cells in a population ofcells to which the gRNA molecule was not introduced or a population ofits progeny, e.g., its erythroid progeny, e.g., its red blood cellprogeny. In embodiments, said cell or its progeny, e.g., its erythroidprogeny, e.g., its red blood cell progeny, produces at least about 6picograms (e.g., at least about 7 picograms, at least about 8 picograms,at least about 9 picograms, at least about 10 picograms, or from about 8to about 9 picograms, or from about 9 to about 10 picograms) fetalhemoglobin per cell.

In an aspect, including in any of the aforementioned aspects andembodiments, the invention provides a gRNA molecule, wherein when aCRISPR system (e.g., an RNP as described herein) including the gRNAmolecule is introduced into a cell, no off-target indels are formed insaid cell, e.g., no off-target indels are formed outside of the HBG1and/or HBG2 promoter regions (e.g., within a gene, e.g., a coding regionof a gene), e.g., as detectible by next generation sequencing and/or anucleotide insertional assay.

In an aspect, including in any of the aforementioned aspects andembodiments, the invention provides a gRNA molecule, wherein when aCRISPR system (e.g., an RNP as described herein) including the gRNAmolecule is introduced into a population of cells, no off-target indel,e.g., no off-target indel outside of the HBG1 and/or HBG2 promoterregions (e.g., within a gene, e.g., a coding region of a gene), isdetected in more than about 5%, e.g., more than about 1%, e.g., morethan about 0.1%, e.g., more than about 0.01%, of the cells of thepopulation of cells, e.g., as detectible by next generation sequencingand/or a nucleotide insertional assay.

In an aspect, including of any of the aforementioned aspects andembodiments, the cell is (or population of cells includes) a mammalian,primate, or human cell, e.g., is a human cell, e.g., the cell is (orpopulation of cells includes) an HSPC, e.g., the HSPC is CD34+, e.g.,the HSPC is CD34+CD90+. In embodiments, the cell is autologous withrespect to a patient to be administered said cell. In other embodiments,the cell is allogeneic with respect to a patient to be administered saidcell.

In an aspect, the gRNA molecules, genome editing systems (e.g., CRISPRsystems), and/or methods described herein relate to cells, e.g., asdescribed herein, that include or result in one or more of the followingproperties:

(a) at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98% or at leastabout 99% of the cells of a population of cells described hereincomprise an indel at or near a genomic DNA sequence complementary to thetargeting domain of a gRNA molecule described herein, optionally whereinthe indel is selected from an indel listed in Table 2-7, optionallywherein no cell of the population comprises a deletion of a nucleotidedisposed between 5,250,092 and 5,249,833, − strand (hg38);

(b) a cell (e.g., population of cells) described herein is capable ofdifferentiating into a differentiated cell of an erythroid lineage(e.g., a red blood cell), and wherein said differentiated cell exhibitsan increased level of fetal hemoglobin, e.g., relative to an unalteredcell (e.g., population of cells);

(c) a population of cells described herein is capable of differentiatinginto a population of differentiated cells, e.g., a population of cellsof an erythroid lineage (e.g., a population of red blood cells), andwherein said population of differentiated cells has an increasedpercentage of F cells (e.g., at least about 15%, at least about 20%, atleast about 25%, at least about 30%, or at least about 40% higherpercentage of F cells) e.g., relative to a population of unalteredcells;

(d) a cell (e.g., population of cells) described herein is capable ofdifferentiating into a differentiated cell, e.g., a cell of an erythroidlineage (e.g., a red blood cell), and wherein said differentiated cell(e.g., population of differentiated cells) produces at least about 6picograms (e.g., at least about 7 picograms, at least about 8 picograms,at least about 9 picograms, at least about 10 picograms, or from about 8to about 9 picograms, or from about 9 to about 10 picograms) fetalhemoglobin per cell;

(e) no off-target indels are formed in a cell described herein, e.g., nooff-target indels are formed outside of the HBG1 and/or HBG2 promoterregions (e.g., within a gene, e.g., a coding region of a gene), e.g., asdetectible by next generation sequencing and/or a nucleotide insertionalassay;

(f) no off-target indel, e.g., no off-target indel outside of the HBG1and/or HBG2 promoter regions (e.g., within a gene, e.g., a coding regionof a gene), is detected in more than about 5%, e.g., more than about 1%,e.g., more than about 0.1%, e.g., more than about 0.01%, of the cells ofa population of cells described herein, e.g., as detectible by nextgeneration sequencing and/or a nucleotide insertional assay;

(g) a cell described herein or its progeny is detectible, e.g.,detectible in the bone marrow or detectible in the peripheral blood, ina patient to which it is transplanted at more than 16 weeks, more than20 weeks or more than 24 weeks after transplantation, optionally asdetected by detecting an indel at or near a genomic DNA sequencecomplementary to the targeting domain of a gRNA molecule of any ofclaims 1-22, optionally wherein the indel is selected from an indellisted in Table 2-7, optionally wherein the indel is a large deletionindel;

(h) a population of cells described herein is capable of differentiatinginto a population of differentiated cells, e.g., a population of cellsof an erythroid lineage (e.g., a population of red blood cells), andwherein said population of differentiated cells includes a reducedpercentage of sickle cells (e.g., at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, or at least about 90% lower percentage of sickle cells) e.g.,relative to a population of unaltered cells; and/or

(i) a cell or population of cells described herein is capable ofdifferentiating into a population of differentiated cells, e.g., apopulation of cells of an erythroid lineage (e.g., a population of redblood cells), and wherein said population of differentiated cellsincludes cells which produce a reduced level (e.g., at least about 15%,at least about 20%, at least about 25%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, or at least about 90% lower level) of sicklehemoglobin (HbS), e.g., relative to a population of unaltered cells.

In an aspect, the invention provides a composition including:

1) one or more gRNA molecules (including a first gRNA molecule)described herein, e.g., of any of the aforementioned gRNA aspects andembodiments, and a Cas9 molecule, e.g., described herein;

2) one or more gRNA molecules (including a first gRNA molecule)described herein, e.g., of any of the aforementioned gRNA aspects andembodiments, and nucleic acid encoding a Cas9 molecule, e.g., describedherein;

3) nucleic acid encoding one or more gRNA molecules (including a firstgRNA molecule) described herein, e.g., of any of the aforementioned gRNAaspects and embodiments, and a Cas9 molecule, e.g., described herein;

4) nucleic acid encoding one or more gRNA molecules (including a firstgRNA molecule) described herein, e.g., of any of the aforementioned gRNAaspects and embodiments, and nucleic acid encoding a Cas9 molecule,e.g., described herein; or

5) any of 1) to 4), above, and a template nucleic acid; or

6) any of 1) to 4) above, and nucleic acid including sequence encoding atemplate nucleic acid.

In an aspect, the invention provides a composition including a firstgRNA molecule described herein, e.g., of any of the aforementioned gRNAaspects and embodiments, further including a Cas9 molecule, e.g.,described herein, e.g., wherein the Cas9 molecule is an active orinactive s. pyogenes Cas9, for example, wherein the Cas9 moleculeincludes SEQ ID NO: 205. In aspects, the Cas9 molecule includes, e.g.,consists of: (a) SEQ ID NO: 233; (b) SEQ ID NO: 234; (c) SEQ ID NO: 235;(d) SEQ ID NO: 236; (e) SEQ ID NO: 237; (f) SEQ ID NO: 238; (g) SEQ IDNO: 239; (h) SEQ ID NO: 240; (i) SEQ ID NO: 241; (j) SEQ ID NO: 242; (k)SEQ ID NO: 243 or (l) SEQ ID NO: 244.

In an aspect, including in any of the aforementioned composition aspectsand embodiments, the first gRNA molecule and Cas9 molecule are presentin a ribonuclear protein complex (RNP).

In an aspect, including in any of the aforementioned composition aspectsand embodiments, the invention provides a composition further includinga second gRNA molecule; a second gRNA molecule and a third gRNAmolecule; or a second gRNA molecule, optionally, a third gRNA molecule,and, optionally, a fourth gRNA molecule, wherein the second gRNAmolecule, the optional third gRNA molecule, and the optional fourth gRNAmolecule are a gRNA molecule described herein, e.g., are a gRNA moleculeof any of the aforementioned gRNA molecule aspects and embodiments, andwherein each gRNA molecule of the composition is complementary to adifferent target sequence. In embodiments, two or more of the first gRNAmolecule, the second gRNA molecule, the optional third gRNA molecule,and the optional fourth gRNA molecule are complementary to targetsequences within the same gene or region. In embodiments, the first gRNAmolecule, the second gRNA molecule, the optional third gRNA molecule,and the optional fourth gRNA molecule are complementary to targetsequences not more than 6000 nucleotides, not more than 5000nucleotides, not more than 500, not more than 400 nucleotides, not morethan 300, not more than 200 nucleotides, not more than 100 nucleotides,not more than 90 nucleotides, not more than 80 nucleotides, not morethan 70 nucleotides, not more than 60 nucleotides, not more than 50nucleotides, not more than 40 nucleotides, not more than 30 nucleotides,not more than 20 nucleotides or not more than 10 nucleotides apart. Inembodiments, two or more of the first gRNA molecule, the second gRNAmolecule, the optional third gRNA molecule, and the optional fourth gRNAmolecule include at least one gRNA molecule which includes a targetingdomain complementary to a target sequence of an HBG1 promoter region andat least one gRNA molecule which includes a targeting domaincomplementary to a target sequence of an HBG2 promoter region. In anaspect, including in any of the aforementioned composition aspects andembodiments, the composition includes (e.g., consists of) a first gRNAmolecule and a second gRNA molecule, wherein the first gRNA molecule andsecond gRNA molecule are: (a) independently selected and target anondeletional HPFH region, e.g., described herein, and are complementaryto different target sequences; (b) independently selected from the gRNAmolecules of Table 1, and are complementary to different targetsequences; c) independently selected from the gRNA molecules of Table 2,and are complementary to different target sequences; or (d)independently selected from the gRNA molecules of Table 3a and arecomplementary to different target sequences, (e) independently selectedfrom the gRNA molecules of Table 3b and are complementary to differenttarget sequences; or (f) independently selected from the gRNA moleculesof any of the aforementioned aspects and embodiments, and arecomplementary to different target sequences.

In an aspect, including in any of the aforementioned composition aspectsand embodiments, the composition includes a first gRNA molecule and asecond gRNA molecule, wherein:

a) the first gRNA molecule is complementary to a target sequenceincluding at least 1 nucleotide (e.g., including 20 consecutivenucleotides) within:

-   -   i) Chr11:5,249,833 to Chr11:5,250,237 (hg38);    -   ii) Chr11:5,250,094-5,250,237 (hg38);    -   iii) Chr11:5,249,833-5,249,927 (hg38); or    -   iv) Chr11:5,250,139-5,250,237 (hg38);

b) the second gRNA molecule is complementary to a target sequenceincluding at least 1 nucleotide (e.g., comprising 20 consecutivenucleotides) within:

-   -   i) Chr11:5,254,738 to Chr11:5,255,164 (hg38);    -   ii) Chr11:5,255,022-5,255,164 (hg38); or    -   iii) Chr11:5,254,738-5,254,851 (hg38).

In an aspect, with respect to the gRNA molecule components of thecomposition, the composition consists of a first gRNA molecule and asecond gRNA molecule.

In an aspect, including in any of the aforementioned composition aspectsand embodiments, each of said gRNA molecules is in a ribonuclear proteincomplex (RNP) with a Cas9 molecule, e.g., described herein.

In an aspect, including in any of the aforementioned composition aspectsand embodiments, the composition includes a template nucleic acid,wherein the template nucleic acid includes a nucleotide that correspondsto a nucleotide at or near the target sequence of the first gRNAmolecule. In embodiments, the template nucleic acid includes nucleicacid encoding: (a) human beta globin, e.g., human beta globin includingone or more of the mutations G16D, E22A and T87Q, or fragment thereof;or (b) human gamma globin, or fragment thereof.

In an aspect, including in any of the aforementioned composition aspectsand embodiments, the composition is formulated in a medium suitable forelectroporation.

In an aspect, including in any of the aforementioned composition aspectsand embodiments, each of said gRNA molecules of said composition is in aRNP with a Cas9 molecule described herein, and wherein each of said RNPis at a concentration of less than about 10 uM, e.g., less than about 3uM, e.g., less than about 1 uM, e.g., less than about 0.5 uM, e.g., lessthan about 0.3 uM, e.g., less than about 0.1 uM. In embodiments, the RNPis at a concentration of about 1 uM. In embodiments, the RNP is at aconcentration of about 2 uM. In embodiments, said concentration is theconcentration of RNP in a composition comprising the cells, e.g., asdescribed herein, optionally wherein the composition comprising thecells and the RNP is suitable for electroporation.

In an aspect, the invention provides a nucleic acid sequence thatencodes one or more gRNA molecules described herein, e.g., of any of theaforementioned gRNA molecule aspects and embodiments. In embodiments,the nucleic acid includes a promoter operably linked to the sequencethat encodes the one or more gRNA molecules, for example, the promoteris a promoter recognized by an RNA polymerase II or RNA polymerase III,or, for example, the promoter is a U6 promoter or an HI promoter.

In an aspect, including in any of the aforementioned nucleic acidaspects and embodiments, the nucleic acid further encodes a Cas9molecule, for example, a Cas9 molecule that includes, e.g., consists of,any of SEQ ID NO: 205, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235,SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ IDNO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243 or SEQ ID NO:244. In embodiments, said nucleic acid includes a promoter operablylinked to the sequence that encodes a Cas9 molecule, for example, anEF-1 promoter, a CMV IE gene promoter, an EF-1α promoter, an ubiquitin Cpromoter, or a phosphoglycerate kinase (PGK) promoter.

In an aspect, the invention provides a vector including the nucleic acidof any of the aforementioned nucleic acid aspects and embodiments. Inembodiments, the vector is selected from the group consisting of alentiviral vector, an adenoviral vector, an adeno-associated viral (AAV)vector, a herpes simplex virus (HSV) vector, a plasmid, a minicircle, ananoplasmid, and an RNA vector.

In an aspect, the invention provides a method of altering a cell (e.g.,a population of cells), (e.g., altering the structure (e.g., sequence)of nucleic acid) at or near a target sequence within said cell,including contacting (e.g., introducing into) said cell (e.g.,population of cells) with:

1) one or more gRNA molecules described herein (e.g., of any of theaforementioned gRNA molecule aspects and embodiments) and a Cas9molecule, e.g., described herein;

2) one or more gRNA molecules described herein (e.g., of any of theaforementioned gRNA molecule aspects and embodiments) and nucleic acidencoding a Cas9 molecule, e.g., described herein;

3) nucleic acid encoding one or more gRNA molecules described herein(e.g., of any of the aforementioned gRNA molecule aspects andembodiments) and a Cas9 molecule, e.g., described herein;

4) nucleic acid encoding one or more gRNA molecules described herein(e.g., of any of the aforementioned gRNA molecule aspects andembodiments) and nucleic acid encoding a Cas9 molecule, e.g., describedherein;

5) any of 1) to 4), above, and a template nucleic acid;

6) any of 1) to 4) above, and nucleic acid including sequence encoding atemplate nucleic acid;

7) a composition described herein, e.g., a composition of any of theaforementioned composition aspects and embodiments; or

8) a vector described herein, e.g., a vector of any of theaforementioned vector aspects and embodiments.

In an aspect, including in any of the aforementioned method aspects andembodiments, the gRNA molecule or nucleic acid encoding the gRNAmolecule, and the Cas9 molecule or nucleic acid encoding the Cas9molecule, are formulated in a single composition. In another aspect, thegRNA molecule or nucleic acid encoding the gRNA molecule, and the Cas9molecule or nucleic acid encoding the Cas9 molecule, are formulated inmore than one composition. In an aspect, the more than one compositionare delivered simultaneously or sequentially.

In an aspect of the methods described herein, including in any of theaforementioned method aspects and embodiments, the cell is an animalcell, for example, the cell is a mammalian, primate, or human cell, forexample, the cell is a hematopoietic stem or progenitor cell (HSPC)(e.g., a population of HSPCs), for example, the cell is a CD34+ cell,for example, the cell is a CD34+CD90+ cell. In embodiments of themethods described herein, the cell is disposed in a compositionincluding a population of cells that has been enriched for CD34+ cells.In embodiments of the methods described herein, the cell (e.g.population of cells) has been isolated from bone marrow, mobilizedperipheral blood, or umbilical cord blood. In embodiments of the methodsdescribed herein, the cell is autologous or allogeneic, e.g.,autologous, with respect to a patient to be administered said cell.

In an aspect of the methods described herein, including in any of theaforementioned method aspects and embodiments, a) the altering resultsin an indel at or near a genomic DNA sequence complementary to thetargeting domain of the one or more gRNA molecules; orb) the alteringresults in a deletion including sequence, e.g., substantially all thesequence, between a sequence complementary to the targeting domain ofthe one or more gRNA molecules (e.g., at least 90% complementary to thegRNA targeting domain, e.g., fully complementary to the gRNA targetingdomain) in the HBG1 promoter region and a sequence complementary to thetargeting domain of the one or more gRNA molecules (e.g., at least 90%complementary to the gRNA targeting domain, e.g., fully complementary tothe gRNA targeting domain) in the HBG2 promoter region. In aspects ofthe method, the indel is an insertion or deletion of less than about 40nucleotides, e.g., less than 30 nucleotides, e.g., less than 20nucleotides, e.g., less than 10 nucleotides, for example, is a singlenucleotide deletion.

In an aspect of the methods described herein, including in any of theaforementioned method aspects and embodiments, the method results in apopulation of cells wherein at least about 15%, e.g., at least about17%, e.g., at least about 20%, e.g., at least about 30%, e.g., at leastabout 40%, e.g., at least about 50%, e.g., at least about 55%, e.g., atleast about 60%, e.g., at least about 70%, e.g., at least about 75% ofthe population have been altered, e.g., include an indel.

In an aspect of the methods described herein, including in any of theaforementioned method aspects and embodiments, the altering results in acell (e.g., population of cells) that is capable of differentiating intoa differentiated cell of an erythroid lineage (e.g., a red blood cell),and wherein said differentiated cell exhibits an increased level offetal hemoglobin, e.g., relative to an unaltered cell (e.g., populationof cells).

In an aspect of the methods described herein, including in any of theaforementioned method aspects and embodiments, the altering results in apopulation of cells that is capable of differentiating into a populationof differentiated cells, e.g., a population of cells of an erythroidlineage (e.g., a population of red blood cells), and wherein saidpopulation of differentiated cells has an increased percentage of Fcells (e.g., at least about 15%, at least about 20%, at least about 25%,at least about 30%, or at least about 40% higher percentage of F cells)e.g., relative to a population of unaltered cells.

In an aspect of the methods described herein, including in any of theaforementioned method aspects and embodiments, the altering results in acell that is capable of differentiating into a differentiated cell,e.g., a cell of an erythroid lineage (e.g., a red blood cell), andwherein said differentiated cell produces at least about 6 picograms(e.g., at least about 7 picograms, at least about 8 picograms, at leastabout 9 picograms, at least about 10 picograms, or from about 8 to about9 picograms, or from about 9 to about 10 picograms) fetal hemoglobin percell.

In an aspect, the invention provides a cell, altered by a methoddescribed herein, for example, a method of any of the aforementionedmethod aspects and embodiments.

In an aspect, the invention provides a cell, obtainable by a methoddescribed herein, for example, a method of any of the aforementionedmethod aspects and embodiments.

In an aspect, the invention provides a cell, including a first gRNAmolecule described herein, e.g., of any of the aforementioned gRNAmolecule aspects or embodiments, or a composition described herein,e.g., of any of the aforementioned composition aspects or embodiments, anucleic acid described herein, e.g., of any of the aforementionednucleic acid aspects or embodiments, or a vector described herein, e.g.,of any of the aforementioned vector aspects or embodiments.

In an aspect of the cell described herein, including in any of theaforementioned cell aspects and embodiments, the cell further includes aCas9 molecule, e.g., described herein, e.g., a Cas9 molecule thatincludes any one of SEQ ID NO: 205, SEQ ID NO: 233, SEQ ID NO: 234, SEQID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO:239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243 orSEQ ID NO: 244.

In an aspect of the cell described herein, including in any of theaforementioned cell aspects and embodiments, the cell includes, hasincluded, or will include a second gRNA molecule described herein, e.g.,of any of the aforementioned gRNA molecule aspects or embodiments, ornucleic acid encoding said gRNA molecule, wherein the first gRNAmolecule and second gRNA molecule include nonidentical targetingdomains.

In an aspect of the cell described herein, including in any of theaforementioned cell aspects and embodiments, expression of fetalhemoglobin is increased in said cell or its progeny (e.g., its erythroidprogeny, e.g., its red blood cell progeny) relative to a cell or itsprogeny of the same cell type that has not been modified to include agRNA molecule.

In an aspect of the cell described herein, including in any of theaforementioned cell aspects and embodiments, the cell is capable ofdifferentiating into a differentiated cell, e.g., a cell of an erythroidlineage (e.g., a red blood cell), and wherein said differentiated cellexhibits an increased level of fetal hemoglobin, e.g., relative to acell of the same type that has not been modified to include a gRNAmolecule.

In an aspect of the cell described herein, including in any of theaforementioned cell aspects and embodiments, the differentiated cell(e.g., cell of an erythroid lineage, e.g., red blood cell) produces atleast about 6 picograms (e.g., at least about 7 picograms, at leastabout 8 picograms, at least about 9 picograms, at least about 10picograms, or from about 8 to about 9 picograms, or from about 9 toabout 10 picograms) fetal hemoglobin, e.g., relative to a differentiatedcell of the same type that has not been modified to include a gRNAmolecule.

In an aspect of the cell described herein, including in any of theaforementioned cell aspects and embodiments, the cell has beencontacted, e.g., contacted ex vivo, with a stem cell expander, forexample, a stem cell expander selected from: a)(1r,4r)—N¹-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamine;b) methyl4-(3-piperidin-1-ylpropylamino)-9H-pyrimido[4,5-b]indole-7-carboxy late;c)4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol;d)(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol;ore) combinations thereof (e.g., a combination of(1r,4r)—N¹-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamineand(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol).In embodiments, the stem cell expander is(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol.

In an aspect of the cell described herein, including in any of theaforementioned cell aspects and embodiments, the cell includes: a) anindel at or near a genomic DNA sequence complementary to the targetingdomain of a gRNA molecule described herein, e.g., of any of theaforementioned gRNA molecule aspects or embodiments; orb) a deletionincluding sequence, e.g., substantially all the sequence, between asequence complementary to the targeting domain of a gRNA moleculedescribed herein, e.g., of any of the aforementioned gRNA moleculeaspects or embodiments (e.g., at least 90% complementary to the gRNAtargeting domain, e.g., fully complementary to the gRNA targetingdomain) in the HBG1 promoter region and a sequence complementary to thetargeting domain of a gRNA molecule described herein, e.g., of any ofthe aforementioned gRNA molecule aspects or embodiments (e.g., at least90% complementary to the gRNA targeting domain, e.g., fullycomplementary to the gRNA targeting domain) in the HBG2 promoter region.In an aspect, the indel is an insertion or deletion of less than about40 nucleotides, e.g., less than 30 nucleotides, e.g., less than 20nucleotides, e.g., less than 10 nucleotides, for example, the indel is asingle nucleotide deletion.

In an aspect of the cell described herein, including in any of theaforementioned cell aspects and embodiments, the cell is an animal cell,for example, the cell is a mammalian, a primate, or a human cell. In anaspect, the cell is a hematopoietic stem or progenitor cell (HSPC)(e.g., a population of HSPCs), e.g., the cell is a CD34+ cell, e.g., thecell is a CD34+CD90+ cell. In embodiments, the cell (e.g. population ofcells) has been isolated from bone marrow, mobilized peripheral blood,or umbilical cord blood. In embodiments, the cell is autologous withrespect to a patient to be administered said cell. In embodiments, thecell the cell is allogeneic with respect to a patient to be administeredsaid cell.

In an aspect, the invention provides a population of cells describedherein, e.g., a population of cells that include a cell describedherein, e.g., a cell of any of the aforementioned cell aspects andembodiments. In aspects, the invention provides a population of cells,wherein at least about 50%, e.g., at least about 60%, e.g., at leastabout 70%, e.g., at least about 80%, e.g., at least about 90% (e.g., atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99%) of the cells of the population are a celldescribed herein, e.g., a cell of any of the aforementioned cell aspectsand embodiments. In aspects, the population of cells (e.g., a cell ofthe population of cells) is capable of differentiating into a populationof differentiated cells, e.g., a population of cells of an erythroidlineage (e.g., a population of red blood cells), and wherein saidpopulation of differentiated cells has an increased percentage of Fcells (e.g., at least about 15%, at least about 17%, at least about 20%,at least about 25%, at least about 30%, or at least about 40% higherpercentage of F cells) e.g., relative to a population of unmodifiedcells of the same type. In aspects, the F cells of the population ofdifferentiated cells produce an average of at least about 6 picograms(e.g., at least about 7 picograms, at least about 8 picograms, at leastabout 9 picograms, at least about 10 picograms, or from about 8 to about9 picograms, or from about 9 to about 10 picograms) fetal hemoglobin percell.

In an aspect, including in any of the aforementioned population of cellaspects and embodiments, the invention provides population of cells,including: 1) at least 1e6 CD34+ cells/kg body weight of the patient towhom the cells are to be administered; 2) at least 2e6 CD34+ cells/kgbody weight of the patient to whom the cells are to be administered; 3)at least 3e6 CD34+ cells/kg body weight of the patient to whom the cellsare to be administered; 4) at least 4e6 CD34+ cells/kg body weight ofthe patient to whom the cells are to be administered; or 5) from 2e6 to10e6 CD34+ cells/kg body weight of the patient to whom the cells are tobe administered. In embodiments, at least about 40%, e.g., at leastabout 50%, (e.g., at least about 60%, at least about 70%, at least about80%, or at least about 90%) of the cells of the population are CD34+cells. In embodiments, at least about 5%, e.g., at least about 10%,e.g., at least about 15%, e.g., at least about 20%, e.g., at least about30% of the cells of the population are CD34+CD90+ cells. In embodiments,the population of cells is derived from umbilical cord blood, peripheralblood (e.g., mobilized peripheral blood), or bone marrow, e.g., isderived from bone marrow. In embodiments, the population of cellsincludes, e.g., consists of, mammalian cells, e.g., human cells. Inembodiments, the population of cells is autologous relative to a patientto which it is to be administered. In other embodiments, the populationof cells is allogeneic relative to a patient to which it is to beadministered.

In an aspect, the invention provides a composition including a celldescribed herein, e.g., a cell of any of the aforementioned cell aspectsand embodiments, or a population of cells described herein, e.g., apopulation of cells of any of the aforementioned population of cellaspects and embodiments. In an aspect, the composition includes apharmaceutically acceptable medium, e.g., a pharmaceutically acceptablemedium suitable for cryopreservation.

In an aspect, the invention provides a method of treating ahemoglobinopathy, including administering to a patient a cell describedherein, e.g., a cell of any of the aforementioned cell aspects andembodiments, a population of cells described herein, e.g., a populationof cells of any of the aforementioned population of cell aspects andembodiments, or a composition described herein, e.g., a composition ofany of the aforementioned composition aspects and embodiments.

In an aspect, the invention provides a method of increasing fetalhemoglobin expression in a mammal, including administering to a patienta cell described herein, e.g., a cell of any of the aforementioned cellaspects and embodiments, a population of cells described herein, e.g., apopulation of cells of any of the aforementioned population of cellaspects and embodiments, or a composition described herein, e.g., acomposition of any of the aforementioned composition aspects andembodiments. In aspects, the hemoglobinopathy is beta-thalassemia. Inaspects, the hemoglobinopathy is sickle cell disease.

In an aspect, the invention provides a method of preparing a cell (e.g.,a population of cells) including:

(a) providing a cell (e.g., a population of cells) (e.g., a HSPC (e.g.,a population of HSPCs));

(b) culturing said cell (e.g., said population of cells) ex vivo in acell culture medium including a stem cell expander; and

(c) introducing into said cell a first gRNA molecule, e.g., describedherein, e.g., a first gRNA molecule of any of the aforementioned gRNAmolecule aspects and embodiments; a nucleic acid molecule encoding afirst gRNA molecule; a composition described herein, e.g., a compositionof any of the aforementioned composition aspects and embodiments; or avector described herein, e.g., a vector of any of the aforementionedaspects and embodiments. In aspects of the method, after saidintroducing of step (c), said cell (e.g., population of cells) iscapable of differentiating into a differentiated cell (e.g., populationof differentiated cells), e.g., a cell of an erythroid lineage (e.g.,population of cells of an erythroid lineage), e.g., a red blood cell(e.g., a population of red blood cells), and wherein said differentiatedcell (e.g., population of differentiated cells) produces increased fetalhemoglobin, e.g., relative to the same cell which has not been subjectedto step (c). In aspects of the method, the stem cell expander is: a)(1r,4r)—N1-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamine;b) methyl4-(3-piperidin-1-ylpropylamino)-9H-pyrimido[4,5-b]indole-7-carboxylate;c)4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol;d)(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol;ore) combinations thereof (e.g., a combination of(1r,4r)—N1-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamineand(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol).In embodiments, the stem cell expander is(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol.In aspects, the cell culture medium includes thrombopoietin (Tpo), Flt3ligand (Flt-3L), and human stem cell factor (SCF). In aspects, the cellculture medium further includes human interleukin-6 (IL-6). In aspects,the cell culture medium includes thrombopoietin (Tpo), Flt3 ligand(Flt-3L), and human stem cell factor (SCF) each at a concentrationranging from about 10 ng/mL to about 1000 ng/mL, for example, each at aconcentration of about 50 ng/mL, for example, each at a concentration of50 ng/mL. In aspects, the cell culture medium includes humaninterleukin-6 (IL-6) at a concentration ranging from about 10 ng/mL toabout 1000 ng/mL, for example, at a concentration of about 50 ng/mL, forexample, at a concentration of 50 ng/mL. In aspects, the cell culturemedium includes a stem cell expander at a concentration ranging fromabout 1 nM to about 1 mM, for example, at a concentration ranging fromabout 1 uM to about 100 nM, for example, at a concentration ranging fromabout 500 nM to about 750 nM. In aspects, the cell culture mediumincludes a stem cell expander at a concentration of about 500 nM, e.g.,at a concentration of 500 nM. In aspects, the cell culture mediumincludes a stem cell expander at a concentration of about 750 nM, e.g.,at a concentration of 750 nM.

In aspects of the method of preparing a cell (e.g., a population ofcells), the culturing of step (b) includes a period of culturing beforethe introducing of step (c), for example, the period of culturing beforethe introducing of step (c) is at least 12 hours, e.g., is for a periodof about 1 day to about 12 days, e.g., is for a period of about 1 day toabout 6 days, e.g., is for a period of about 1 day to about 3 days,e.g., is for a period of about 1 day to about 2 days, e.g., is for aperiod of about 2 days. In aspects of the method of preparing a cell(e.g., a population of cells), including in any of the aforementionedaspects and embodiments of the method, the culturing of step (b)includes a period of culturing after the introducing of step (c), forexample, the period of culturing after the introducing of step (c) is atleast 12 hours, e.g., is for a period of about 1 day to about 12 days,e.g., is for a period of about 1 day to about 6 days, e.g., is for aperiod of about 2 days to about 4 days, e.g., is for a period of about 2days or is for a period of about 3 days or is for a period of about 4days. In aspects of the method of preparing a cell (e.g., a populationof cells), including in any of the aforementioned aspects andembodiments of the method, the population of cells is expanded at least4-fold, e.g., at least 5-fold, e.g, at least 10-fold, e.g., relative tocells which are not cultured according to step (b).

In aspects of the method of preparing a cell (e.g., a population ofcells), including in any of the aforementioned aspects and embodimentsof the method, the introducing of step (c) includes an electroporation.In aspects, the electroporation includes 1 to 5 pulses, e.g., 1 pulse,and wherein each pulse is at a pulse voltage ranging from 700 volts to2000 volts and has a pulse duration ranging from 10 ms to 100 ms. Inaspects, the electroporation includes, e.g., consists of, 1 pulse. Inaspects, the pulse (or more than one pulse) voltage ranges from 1500 to1900 volts, e.g., is 1700 volts. In aspects, the pulse duration of theone pulse or more than one pulse ranges from 10 ms to 40 ms, e.g., is 20ms.

In aspects of the method of preparing a cell (e.g., a population ofcells), including in any of the aforementioned aspects and embodimentsof the method, the cell (e.g., population of cells) provided in step (a)is a human cell (e.g., a population of human cells). In aspects of themethod of preparing a cell (e.g., a population of cells), including inany of the aforementioned aspects and embodiments of the method, thecell (e.g., population of cells) provided in step (a) is isolated frombone marrow, peripheral blood (e.g., mobilized peripheral blood) orumbilical cord blood. In aspects of the method of preparing a cell(e.g., a population of cells), including in any of the aforementionedaspects and embodiments of the method, the cell (e.g., population ofcells) provided in step (a) is isolated from bone marrow, e.g., isisolated from bone marrow of a patient suffering from ahemoglobinopathy.

In aspects of the method of preparing a cell (e.g., a population ofcells), including in any of the aforementioned aspects and embodimentsof the method, the population of cells provided in step (a) is enrichedfor CD34+ cells.

In aspects of the method of preparing a cell (e.g., a population ofcells), including in any of the aforementioned aspects and embodimentsof the method, subsequent to the introducing of step (c), the cell(e.g., population of cells) is cryopreserved.

In aspects of the method of preparing a cell (e.g., a population ofcells), including in any of the aforementioned aspects and embodimentsof the method, subsequent to the introducing of step (c), the cell(e.g., population of cells) includes: a) an indel at or near a genomicDNA sequence complementary to the targeting domain of the first gRNAmolecule; orb) a deletion including sequence, e.g., substantially allthe sequence, between a sequence complementary to the targeting domainof the first gRNA molecule (e.g., at least 90% complementary to the gRNAtargeting domain, e.g., fully complementary to the gRNA targetingdomain) in the HBG1 promoter region and a sequence complementary to thetargeting domain of the first gRNA molecule (e.g., at least 90%complementary to the gRNA targeting domain, e.g., fully complementary tothe gRNA targeting domain) in the HBG2 promoter region.

In aspects of the method of preparing a cell (e.g., a population ofcells), including in any of the aforementioned aspects and embodimentsof the method, after the introducing of step (c), at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 96%, atleast about 97%, at least about 98% or at least about 99% of the cellsof the population of cells include an indel at or near a genomic DNAsequence complementary to the targeting domain of the first gRNAmolecule.

In an aspect, the invention provides a cell (e.g., population of cells),obtainable by a method of preparing a cell (e.g., a population of cells)described herein, e.g., described in any of the aforementioned method ofpreparing a cell aspects and embodiments.

In an aspect, the invention provides a method of treating ahemoglobinopathy in a human patient, including administering to a humanpatient a composition including a cell described herein, e.g., a cell ofany of the aforementioned cell aspects and embodiments; or a populationof cells described herein, e.g., a population of cells of any of theaforementioned population of cell aspects and embodiments.

In aspects, the hemoglobinopathy is beta-thalassemia. In aspects, thehemoglobinopathy is sickle cell disease.

In an aspect, the invention provides a method of increasing fetalhemoglobin expression in a human patient, including administering tosaid human patient a composition including a cell described herein,e.g., a cell of any of the aforementioned cell aspects and embodiments;or a population of cells described herein, e.g., a population of cellsof any of the aforementioned population of cell aspects and embodiments.In aspects, the human patients has beta-thalassemia. In aspects, thehuman patient has sickle cell disease.

In aspects of the method of treating a hemoglobinopathy or the method ofincreasing fetal hemoglobin expression, the human patient isadministered a composition including at least about 1e6 cells (e.g.,cells as described herein) per kg body weight of the human patient,e.g., at least about 1e6 CD34+ cells (e.g., cells as described herein)per kg body weight of the human patient. In aspects of the method oftreating a hemoglobinopathy or the method of increasing fetal hemoglobinexpression, the human patient is administered a composition including atleast about 2e6 cells (e.g., cells as described herein) per kg bodyweight of the human patient, e.g., at least about 2e6 CD34+ cells (e.g.,cells as described herein) per kg body weight of the human patient. Inaspects of the method of treating a hemoglobinopathy or the method ofincreasing fetal hemoglobin expression, the human patient isadministered a composition including about 2e6 cells (e.g., cells asdescribed herein) per kg body weight of the human patient, e.g., about2e6 CD34+ cells (e.g., cells as described herein) per kg body weight ofthe human patient. In aspects of the method of treating ahemoglobinopathy or the method of increasing fetal hemoglobinexpression, the human patient is administered a composition including atleast about 3e6 cells (e.g., cells as described herein) per kg bodyweight of the human patient, e.g., at least about 3e6 CD34+ cells (e.g.,cells as described herein) per kg body weight of the human patient. Inaspects of the method of treating a hemoglobinopathy or the method ofincreasing fetal hemoglobin expression, the human patient isadministered a composition including about 3e6 cells (e.g., cells asdescribed herein) per kg body weight of the human patient, e.g., about3e6 CD34+ cells (e.g., cells as described herein) per kg body weight ofthe human patient. In aspects of the method of treating ahemoglobinopathy or the method of increasing fetal hemoglobinexpression, the human patient is administered a composition includingfrom about 2e6 to about 10e6 cells (e.g., cells as described herein) perkg body weight of the human patient, e.g., from about 2e6 to about 10e6CD34+ cells (e.g., cells as described herein) per kg body weight of thehuman patient.

In an aspect, the invention provides: a gRNA molecule described herein,e.g., a gRNA molecule of any of the aforementioned gRNA molecule aspectsand embodiments; a composition described herein, e.g., a composition ofany of the aforementioned composition aspects and embodiments, a nucleicacid described herein, e.g., a nucleic acid of any of the aforementionednucleic acid aspects and embodiments; a vector described herein, e.g., avector of any of the aforementioned vector aspects and embodiments; acell described herein, e.g., a cell of any of the aforementioned cellaspects and embodiments; or a population of cells described herein,e.g., a population of cells of any of the aforementioned population ofcells aspects and embodiments, for use as a medicament.

In an aspect, the invention provides: a gRNA molecule described herein,e.g., a gRNA molecule of any of the aforementioned gRNA molecule aspectsand embodiments; a composition described herein, e.g., a composition ofany of the aforementioned composition aspects and embodiments, a nucleicacid described herein, e.g., a nucleic acid of any of the aforementionednucleic acid aspects and embodiments; a vector described herein, e.g., avector of any of the aforementioned vector aspects and embodiments; acell described herein, e.g., a cell of any of the aforementioned cellaspects and embodiments; or a population of cells described herein,e.g., a population of cells of any of the aforementioned population ofcells aspects and embodiments, for use in the manufacture of amedicament.

In an aspect, the invention provides: a gRNA molecule described herein,e.g., a gRNA molecule of any of the aforementioned gRNA molecule aspectsand embodiments; a composition described herein, e.g., a composition ofany of the aforementioned composition aspects and embodiments, a nucleicacid described herein, e.g., a nucleic acid of any of the aforementionednucleic acid aspects and embodiments; a vector described herein, e.g., avector of any of the aforementioned vector aspects and embodiments; acell described herein, e.g., a cell of any of the aforementioned cellaspects and embodiments; or a population of cells described herein,e.g., a population of cells of any of the aforementioned population ofcells aspects and embodiments, for use in the treatment of a disease.

In an aspect, the invention provides: a gRNA molecule described herein,e.g., a gRNA molecule of any of the aforementioned gRNA molecule aspectsand embodiments; a composition described herein, e.g., a composition ofany of the aforementioned composition aspects and embodiments, a nucleicacid described herein, e.g., a nucleic acid of any of the aforementionednucleic acid aspects and embodiments; a vector described herein, e.g., avector of any of the aforementioned vector aspects and embodiments; acell described herein, e.g., a cell of any of the aforementioned cellaspects and embodiments; or a population of cells described herein,e.g., a population of cells of any of the aforementioned population ofcells aspects and embodiments, for use in the treatment of a disease,wherein the disease is a hemoglobinopathy, for example, beta-thalassemiaor sickle cell disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: HbF induction 7 days after editing. For each gRNA targetsequence tested the percentage of cells with induced HbF expressioncorrected for background levels based on mock transfection, is shown asmean with error bar indicating standard deviation. The gRNA G8 againstexon 2 of BCL11A serves as a positive control. A dotted line at 17%indicates the threshold level chosen for the analysis. Various greyshading, as indicated in the legend relate the degree of HbF inductionto the degree of editing in the HBG1 or HBG2 target loci.

FIG. 2: Editing efficiency at the HBG1 target locus. For each gRNAtested the percentage of indels detected by NGS is shown as mean witherror bar indicating standard deviation. The gRNA G8 against exon 2 ofBCL11A serves as a positive control. Two guides for which no NGS datawas obtained are indicated by arrowheads.

FIG. 3: Editing efficiency at the HBG2 target locus. For each gRNAtested the percentage of indels detected by NGS is shown as mean witherror bar indicating standard deviation. The gRNA G8 against exon 2 ofBCL11A serves as a positive control. Sixteen guides for which no NGSdata was obtained are indicated by arrowheads.

FIG. 4: Overview of the location of high performing gRNA targetsequences (e.g., >17% HbF upregulation at Day 7), known non-deletionalHPFH polymorphisms and transcription factor binding sites in the HBG1promoter area. FIGS. 4A-4D disclose SEQ ID NOS 293-312, respectively, inorder of appearance.

FIG. 5: Overview of the location of high-performing gRNA targetsequences (e.g., >17% HbF upregulation at Day 7), known non-deletionalHPFH polymorphisms and transcription factor binding sites in the HBG2promoter area. FIGS. 5A-5D disclose SEQ ID NOS 313-332, respectively, inorder of appearance.

FIG. 6: Editing efficiency at targeted B2M locus in CD34+ HSPCs bydifferent Cas9 variants, as evaluated by NGS and Flow cytometry.NLS=SV40 NLS; His6 (SEQ ID NO: 247) or His8 (SEQ ID NO: 248) refers to 6(SEQ ID NO: 247) or 8 (SEQ ID NO: 248) histidine residues, respectively;TEV=tobacco etch virus cleavage site; Cas9=wild type S. pyogenesCas9—mutations or variants are as indicated).

FIG. 7: Detection and quantification of HbF positive cells by flowcytometry at 7 (black bars), 14 (light gray bars) or 21 (dark gray bars)days after erythroid differentiation following electroporation of HSPCswith RNPs containing sgRNA of the indicated targeting domain. Thepercentage HbF+ cells for control cultures not treated with sgRNA ateach time point has been subtracted. Mean+standard deviation isindicated (n=2 technical replicates).

FIG. 8: Detection and quantification of HbF positive cells by flowcytometry at 7 (open black bars), 14 (open light gray bars) or 21 (opendark gray bars) days after erythroid differentiation followingelectroporation of HSPCs with RNPs containing sgRNA of the indicatedtargeting domain. The percentage HbF+ cells for control cultures nottreated with sgRNA at each time point has been subtracted. Mean (bar) oftwo independent cell donors is shown, along with value for each donor(circle=first donor, triangle=second donor).

FIG. 9. Visualization of PCR products from the indicated reaction: P1,P2 or P3, as described in the Examples, from cells followingelectroporation with RNPs containing sgRNA of the indicated targetingdomain or control cells not treated with sgRNA. Expected products are asfollows. P1: 7.7 kb for wild type/small indel allele or 4.9 kb inversionallele, 2.8 kb for 4.9 kb deleted allele. P2: 3.8 kb for wild type/smallindel allele, no product for 4.9 kb deletion or 4.9 kb inversion allele.P3: 1.8 kb for 4.9 kb inversion allele, no product for wild type/smallindel allele or 4.9 kb deletion allele. L=DNA reference ladder. *=DNAfrom this band was isolated and subjected to next-generation sequencing.

FIG. 10: Schematic indicating genomic location of primer and probebinding sites for the digital droplet PCR assay to quantify 4.9 kbdeletions. The primers (5.2 kb Fwd and 5.2 kb Rev) and probe (FAM probe)bind to an intergenic site downstream of HBG2 and upstream of HBG1. Theprobe has a second binding site upstream of HBG2, but that region is notbound by the primers. The areas in which the targeting domains in theHBG1 and HBG2 promotors are located are indicated. If the sequenceintervening the two targeting domain regions is deleted, theprimer/probe binding site between HBG1 and HBG2 would be lost.

FIG. 11: Sorting scheme for HSPC subpopulations for the cell samplefollowing electroporation with RNPs containing sgRNA of the GCR-0067targeting domain. Dot plots of cellular fluorescence followingimmunostaining targeted to the indicated cell surface marker are shown.The following populations were sorted as shown: P5=CMP(CD34+CD45RA−CD38+), P9=MPP (CD34+CD45RA−CD38−CD90−CD49f−), P10=ST-HSC(CD34+CD45RA−CD38−CD90−CD49f+), P11=LT-HSC(CD34+CD45RA−CD38−CD90+CD49f+). Total CD34+ cells were also sorted (notshown).

FIG. 12: Percent editing of sorted HSPC subpopulations followingelectroporation with RNPs containing sgRNA of the GCR-0067 targetingdomain. HBG1 indel and HBG2 indel indicates the percentage of smallinsertions and deletions identified by next generation sequencing of PCRamplicons at or near the HBG1 or HBG2 promotor region targeting domain,respectively (note that alleles with the previously described 4.9 kbdeletion or inversion are not amplified). HBG1-HBG2 deletion indicatesthe percentage of alleles with the 4.9 kb deletion, as determined by thedigital droplet PCR assay described in the Examples. Total editing is anapproximation calculated by the percentage of HBG1-HBG2 deletion addedto the percentage without HBG1-HBG2 deletion times the percentage HBG2indel.

FIG. 13: FIG. 13A shows the sum of all indels observed at the HBG1 locusin the stated cell type after introduction of sgRNA comprising thetargeting domain of GCR-0067. Indels are arranged with most frequentlyobserved indels at the top of each bar. Not quantified in this assay isthe fraction of cells comprising the large 4.9 kb deletion between HBG1and HBG2 loci. The number within the bar of each of the most prevalentindels indicates the number of nucleotide differences from the wild-typegenomic sequence (− indicates deletion; + indicates insertion). FIG. 13Bshows the sum of all indels observed at the HBG2 locus in the statedcell type after introduction of sgRNA comprising the targeting domain ofGCR-0067. Indels are arranged with most frequently observed indels atthe top of each bar. Not quantified in this assay is the fraction ofcells comprising the large 4.9 kb deletion between HBG1 and HBG2 loci.The number within the bar of each of the most prevalent indels indicatesthe number of nucleotide differences from the wild-type genomic sequence(− indicates deletion; + indicates insertion). CMP=CD34+CD45RA−CD38+cells; MPP=CD34+CD45RA−CD38−CD90−CD49f− cells;ST-HSC=CD34+CD45RA−CD38−CD90−CD49f+ cells; andLT-HSC=CD34+CD45RA−CD38−CD90+CD49f+ cells.

FIG. 14: Percentage of colonies corresponding to the indicated subtype,CFU-GEMM (dark gray bars), CFU-G/M/GM (medium gray bars) or BFU-E/CFU-E(light gray bars), following electroporation with RNPs containing sgRNAof the indicated targeting domain or control cultures not treated withsgRNA. Mean+/−standard deviation is indicated (n=2 independent donors).

FIG. 15: Fold proliferation of total nucleated cells (TNC), CD34positive cells (CD34+) and CD34 and CD90 dual positive cells(CD34+CD90+), as indicated, in medium comprising compound 4, followingelectroporation with RNPs containing sgRNA of the GCR-0067 targetingdomain or control cultures not treated with sgRNA. Mean+/−standarddeviation is indicated (n=2 independent donors). Mean (bar) of threeindependent cell donors is shown, along with value for each donor(square=donor A, triangle=donor B, circle=donor C). Differences betweenedited and control cultures were not significant (ns) by unpaired t-test(GraphPad Prism).

FIG. 16: Representative gating of cellular populations by flowcytometry. Dot plots of cellular fluorescence following immunostainingtargeted to the indicated cell surface markers, or isotype control(isotype), are shown in FIGS. 16A and 16B. Gates indicated with the boldboxes were used to quantify the percentage of the indicated populationand were set to exclude isotype control labeled cells. Only viable cellspre-gated as DAPI negative and within the cellular forward scatter andside scatter gates are shown and are derived from donor C electroporatedwith Cas9 alone.

FIG. 17: Percentage of cells with the indicated cellular cell surfacephenotype, as assessed by flow cytometry, following electroporation withRNPs containing sgRNA of the GCR-0067 targeting domain (black bars) orcontrol cultures not treated with sgRNA (gray bars). Cells were expanded2 days post electroporation of RNP in medium comprising Compound 4, andassessed by flow cytometry as described in FIGS. 16A and 16B.Mean+standard deviation is indicated (n=3 independent donors). Therewere no significant differences between edited and unedited cells for agiven population by unpaired t-test (GraphPad Prism).

FIG. 18A. CE-MS quantification of globin subunits in mock edited HSCsderived from sickle cell disease patient (SCD1) mock edited with Cas9and no sgRNA. After cells are differentiated into erythroid lineage,cells showed normal level of a-globin, no normal b-globin due to sicklehomozygosity, high level of sickle b-globin subunit, and low level offetal g-globin.

FIG. 18B. CE-MS quantification of globin subunits in genome-edited HSPCsderived from sickle cell patients (SCD1). After editing the HSCs,erythroid cells derived from the sample patient demonstrated 40%upregulation of fetal g-globin and a concurrent 50% downregulation ofsickle b-globin subunit.

FIG. 19. Schematic protocol for studying engraftment of gene editedcells. Transplantation of HSCs gene-edited with Cas9 and sgRNAcomprising the targeting domain of CR001128 (sg1128). Five hundredthousand human CD34+ cells were either mock edited with gRNA or geneedited with sg1128, followed by injection into 2 Gy irradiatedNOD.Cg-Prkdc^(scid)Il2rg^(tm1Wjl)/SzJ (NSG) recipients. Mice were bledat 4, 8, 12, 16 weeks and bone marrow cells were harvested at 16 weekspost-transplant.

FIG. 20. Bone marrow reconstitution at 16 weeks post-transplant usingthe experimental protocol shown in FIG. 19.

FIG. 21. Reconstitution of myeloid, B, and T lymphoid cells in theperipheral blood and bone marrow at multiple time points using theexperimental protocol shown in FIG. 19. N=5/group, data show min to max,4 independent experiments.

FIG. 22. Schematic diagram of transplant study to evaluate stem cellfunction of HSCs edited with sgRNAs from the gamma globin promoterregion (sg-G0008, sg-G0051, sg-G0010, sg-G0048, sg-G0067) in comparisonto gRNA from the erythroid-specific enhancer region of the BCL11A gene(sg-G1128; also referred to as sg1128). Cells were left in culture 24hours post electroporation. Culture conditions both before and afterelectroporation were StemSpan SFEM+IL6, SCF, TPO, Flt3L; 750 nM Compound4.

FIG. 23: Human engraftment and lineage analysis over 20 weeks in NSGmice. FIG. 23A) Peripheral blood chimerism over 18 weeks; FIG. 23B)Lineage distribution in the peripheral blood at 18 weeks. Bone marrowanalysis: FIG. 23C) Bone marrow analysis of human cells at week 9; FIG.23D) human CD45+ engraftment and lineage distribution of human cells inbone marrow at week 9; FIG. 23E) human CD45+ engraftment in bone marrowat week 20 post engraftment; FIG. 23F) Lineage distribution of engraftedcells in the bone marrow at 20 weeks.

FIG. 24A. Engraftment efficiency of HSCs edited with sgRNAs withhomology to the gamma globin promote region (sg-G0008, sg-G0051,sg-G0010, sg-G0048, sg-G0067) compared to sgRNA from theerythroid-specific enhancer region of the BCL11A gene (sg1128). Showshuman cell engraftment in NSG mice at 8 weeks following transplantationN=10/group, 3 independent experiments. Graph shows pooled data.

FIG. 24B. Engraftment efficiency of HSCs edited with sgRNAs withhomology to the gamma globin promote region (sg-G0008, sg-G0051,sg-G0010, sg-G0048, sg-G0067) compared to sgRNA from theerythroid-specific enhancer region of the BCL11A gene (sg1128). Showshuman cell engraftment in NSG mice at 20 weeks post-transplant.N=10/group, 3 independent experiments. Graph shows pooled data.

FIG. 25. Multi-lineage reconstitution of gene-edited CD34+ celltransplanted NSG mice. N=10/group, data from one representativeexperiment.

FIG. 26. High editing efficiency was maintained pre- andpost-transplantation “Pre-Xpt”: editing efficiency and indel pattern ofindividual sgRNA in human CD34+ cells as measured by NGS upon editingbut prior to transplantation. “8 wks Post-Xpt”: editing efficiency andindel pattern in human CD34+ cells 8 weeks after bone marrowtransplantation in mice as measured by NGS. “20 wks Post-Xpt”: editingefficiency and indel pattern in human CD34+ cells 20 weeks after bonemarrow transplantation in mice as measured by NGS. Electroporationperformed in triplicate per group, data from one representativeexperiment. As used in relation to this Figure, “indel” refers to sum ofall indels of less than 200 nt; “large deletion” refers to the deletionof sequence between the predicted HBG1 and HBG2 binding sites for eachgRNA.

FIG. 27. NGS analysis of CD34+ cells post-editing with RNPs. sgRNAtargeting specific regions are indicated on the x-axis. FIG. 27A: NGSanalysis of CD34+ cells at day 2 post-electroporation of RNPs. FIG. 27B:NGS analysis of whole bone marrow from NSG mice transplanted with editedbone marrow CD34+ cells at (27B) week 9 post-transplantation FIG. 27C:NGS analysis of whole bone marrow from NSG mice transplanted with editedbone marrow CD34+ cells at week 20 post-transplantation. Insertionindels are indicated in black, deletion indels (excluding largedeletions comprising an excision of the region between binding HBG1 andHBG2 target sequences for each of gRNA sg-G51, sg-G48 and sg-G67) areindicated in grey. Total % editing is represented by the height of thebars. N=10, data are presented as mean±SEM from one independentexperiment.

FIG. 28. Gene-edited, long-term engrafted human HSCs were capable toproducing increased level of HbF upon erythroid cell differentiationFifty thousand human CD34+ cells were sorted from the bone marrow of8-week or 20-week transplanted NSG mice. Sorted cells were seeded intoerythroid differentiation medium for 14-21 days. Mature red blood cellsin culture were assayed for HbF expression and to enumerate the numberof HbF+ cell by flow cytometry. Mock control represents CD34+ cellsedited with Cas9 without gRNA, and transplanted into NSG mice in equalmanner as gene-edited control group. N=10/group, 3 independentexperiments.

FIG. 29. Off-target activity for HBG1 and/or HBG2 guide RNAs wasassessed using an dsDNA oligo-insertion method in Cas9-overexpressingHEK-293 cells. The on-target site (open circle) and the potentialoff-target sites (closed circles) detected are indicated; y-axisindicates frequency of detection. All gRNAs were tested in dgRNA formatwith the targeting domain indicated by the CRxxxxxx identifier.

FIG. 30. Off-target activity for HBG1 and/or HBG2 guide RNAs wasassessed using an dsDNA oligo-insertion method in Cas9-overexpressingHEK-293 cells. The on-target site (open circle) and the potentialoff-target sites (closed circles) detected are indicated; y-axisindicates frequency of detection. gRNAs were tested in either dgRNAformat with the targeting domain indicated by the CRxxxxx identifier, orin sgRNA format with the targeting domain indicated by the Gxxxxxxidentifier.

FIG. 31A. CD34+ cell count of cells derived from mobilized peripheralblood of healthy individuals upon gene-editing. Cells were thawed on day0, cultured for 3 days prior to electroporation on day 3. Enumeration ofCD34+ cell number by ISHAGE over 10 days following electroporation. Twoindependent experiments were performed in duplicates. Total N=5. Graphsshow data from 1 experiment with mean±SEM.

FIG. 31B. CD34+ cell expansion of cells derived from mobilizedperipheral blood of healthy individuals upon gene-editing. Cells werethawed on day 0, cultured for 3 days prior to electroporation on day 3.Expansion of total mononuclear cells at day 3, 7, and 10 upon editing.Two independent experiments were performed in duplicates. Total N=5.Graphs show data from 1 experiment with mean±SEM.

FIG. 31C. CD34+ cell viability of cells derived from mobilizedperipheral blood of healthy individuals upon gene-editing. Cells werethawed on day 0, cultured for 3 days prior to electroporation on day 3.Viability of mononuclear cells at the same time points post-editing. Twoindependent experiments were performed in duplicates. Total N=5. Graphsshow data from 1 experiment with mean±SEM.

FIG. 32A. Editing efficiency of sg1128 and sg0067 in CD34+ cellsmobilized from the peripheral blood of healthy individuals. Shown is thepercentage of INDELs captured by NGS upon editing using sg1128(targeting the BCL11A+58 region of ESH) and sg0067 (targeting the HbG-1and HbG-2 gene cluster). This graph also indicates the total editingefficiency for sg1128 as only small INDELs were generated by this sgRNA.More than five independent experiments were performed in duplicate ortriplicate, n=2-3/experiment.

FIG. 32B. Editing efficiency of sg1128 and sg0067 in CD34+ cellsmobilized from the peripheral blood of healthy individuals. A. Thepercentage of INDELs captured by NGS upon editing using sg0067(targeting the HbG-1 and HbG-2 gene cluster). Shown is the total editingefficiency and editing pattern of sg0067. The editing pattern of sg0067consists of a 5 kb large deletion (denoted by black bars) and smallerINDELs (denoted by grey bars). More than five independent experimentswere performed in duplicate or triplicate, n=2-3/experiment.

FIG. 33. Fold change of g-globin transcript in erythroid cells frompatient samples upon CRISPR knockdown of BCL11A or indel/deletionformation in the HBG1/2 region. CD34+ cells derived from the mobilizedperipheral blood of healthy donors were edited by CRISPR anddifferentiated into the erythroid lineage in vitro as described inprevious procedures. At day 11 of erythroid differentiation, cells wereharvested from culture and subjected to qPCR to measure g-globin andb-globin transcripts, normalizing to GAPDH. Experiment was performedindependently twice, each in duplicate, n=2-3/experiment. Data showmean±SEM of pooled donors from one study.

FIG. 34. Enumeration of HbF+ cells from healthy individuals upon geneediting. CD34+ cells derived from the mobilized peripheral blood ofhealthy donors were edited by CRISPR and differentiated into theerythroid lineage in vitro as described in previous procedures. At day10, of erythroid differentiation, cells were stained with anti-HbF-FITCantibody to enumerate HbF+ cells by flow cytometry. Experiment wasperformed independently twice, each in duplicate, n=2-3/experiment. Datashow average±SD from one study.

FIG. 35A. Expansion and viability of CD34+ cells derived from theperipheral blood of sickle cell disease individuals upon gene-editing.Cells were cultured for 6-10 days prior to electroporation. D0 refers tothe day of electroporation. Shown is the absolute count of CD34+ cellsby ISHAGE over 10 days following electroporation. N=4, data showmean±SEM. 4 independent experiments performed in duplicates. Bars are,from left to right at each time point, mock, sg1128, sg0067.

FIG. 35B. Expansion and viability of CD34+ cells derived from theperipheral blood of sickle cell disease individuals upon gene-editing.Cells were cultured for 6-10 days prior to electroporation. D0 refers tothe day of electroporation. Shown is the percentage of CD34+ cells byISHAGE over 10 days following electroporation. N=4, data show mean±SEM.4 independent experiments performed in duplicates. Bars are, from leftto right at each time point, mock, sg1128, sg0067.

FIG. 35C. Expansion and viability of CD34+ cells derived from theperipheral blood of sickle cell disease individuals upon gene-editing.Cells were cultured for 6-10 days prior to electroporation. D0 refers tothe day of electroporation. Shown is expansion of total mononuclearcells at day 3, 7, and 10 upon editing. N=4, data show mean±SEM. 4independent experiments performed in duplicates. Bars are, from left toright at each time point, mock, sg1128, sg0067.

FIG. 35D. Expansion and viability of CD34+ cells derived from theperipheral blood of sickle cell disease individuals upon gene-editing.Cells were cultured for 6-10 days prior to electroporation. D0 refers tothe day of electroporation. Shown is viability of mononuclear cells atday 3, 7 and 10 post-editing. N=4, data show mean±SEM. 4 independentexperiments performed in duplicates. Bars are, from left to right ateach time point, mock, sg1128, sg0067.

FIG. 36. Editing efficiency of sg1128 and sg0067 in CD34+ cells fromsickle cell disease patient samples. Editing pattern of sg0067 wasdenoted by large deletion (grey) and sum of small indels (black). Datashow mean±SEM of four independent editing experiments performed induplicates with CD34+ cells from four different sickle cell diseasepatients (SCD1-4).

FIG. 37. In vitro multi-lineage differentiation capacity of HSPCs asmeasured by colony-forming unit assay. BFU-E=blast-forming unit,erythroid; CFU-GM=colony-forming unit, granulocyte, monocyte;CFU-GEMM=colony-forming unit, granulocyte, erythroid, monocyte,megakaryocyte. Graph shows three independent experiments from CD34+cells from three different sickle cell disease patients (SCD1-3).Experiments were performed in triplicates. Data represent mean±SEM.

FIG. 38. Fold change of g-globin transcript in erythroid cells frompatient samples upon CRISPR knockdown of BCL11A or indel/deletionformation at g-globin gene cluster. CD34+ cells derived from 3 sicklecell disease patients (SCD1-3) were edited by CRISPR and differentiatedinto the erythroid lineage in vitro as described in the Examples. At day11 of erythroid differentiation, cells were harvested from culture andsubjected to qPCR to measure g-globin and b-globin transcripts,normalizing to GAPDH. Experiment was performed in duplicate. Data showmean±SEM of pooled donors.

FIG. 39. Enumeration of HbF+ cells in sickle cell disease patientsamples upon gene editing. CD34+ cells derived from 3 sickle celldisease patients (SCD1-3) were edited by CRISPR and differentiated intothe erythroid lineage in vitro as described in the Examples. At day 11,14, and 21 of erythroid differentiation, cells were stained withanti-HbF-FITC antibody to enumerate HbF+ cells by flow cytometry.Experiment was performed in duplicate. Data show mean±SEM. Shown at day11 are results from SCD1-3; shown at day 14 are results from SCD-1 andSCD-2; shown at day 21 are results from SCD-2 and SCD-3.

FIG. 40. Measurement of fetal hemoglobin expression in gene-editedsickle cell disease patient samples by flow cytometry. CD34+ cellsderived from 3 sickle cell disease patients (SCD1-3) were edited byCRISPR and differentiated into the erythroid lineage in vitro asdescribed in the Examples. At day 11, 14, and 21 of erythroiddifferentiation, cells were stained with anti-HbF-FITC antibody tomeasure the HbF expression intensity of each cell by flow cytometry.Experiment was performed in duplicate. Data show mean±SEM. MFI=meanfluorescent intensity. Shown at day 11 are results from SCD1-3; shown atday 14 are results from SCD-1 and SCD-2; shown at day 21 are resultsfrom SCD-2 and SCD-3.

FIG. 41. Enumeration of the number of sickle cells versus normal cellsupon CRISPR editing of patient samples. CD34+ cells derived from 3sickle cell disease patients (SCD1-3) were edited by CRISPR anddifferentiated into the erythroid lineage in vitro as described inprevious procedures. At day 21 of erythroid differentiation, cells weresubjected to a % hypoxia chamber for 4 days, fixed, and followed bysingle cell imaging flow cytometry. FIG. 41A (left panel) shows changein the number of sickle cells in gene-edited patient samples asenumerated by single cell imaging. FIG. 41B (right panel) shows changein the number of normal cells after gene editing in patient samples asenumerated by single cell imaging. Three independent experiments wereperformed, with each experiment in duplicate. Forty thousand single cellimages were enumerated from each patient. Graph show mean±SEM frompooled data.

DEFINITIONS

The terms “CRISPR system,” “Cas system” or “CRISPR/Cas system” refer toa set of molecules comprising an RNA-guided nuclease or other effectormolecule and a gRNA molecule that together are necessary and sufficientto direct and effect modification of nucleic acid at a target sequenceby the RNA-guided nuclease or other effector molecule. In oneembodiment, a CRISPR system comprises a gRNA and a Cas protein, e.g., aCas9 protein. Such systems comprising a Cas9 or modified Cas9 moleculeare referred to herein as “Cas9 systems” or “CRISPR/Cas9 systems.” Inone example, the gRNA molecule and Cas molecule may be complexed, toform a ribonuclear protein (RNP) complex.

The terms “guide RNA,” “guide RNA molecule,” “gRNA molecule” or “gRNA”are used interchangeably, and refer to a set of nucleic acid moleculesthat promote the specific directing of a RNA-guided nuclease or othereffector molecule (typically in complex with the gRNA molecule) to atarget sequence. In some embodiments, said directing is accomplishedthrough hybridization of a portion of the gRNA to DNA (e.g., through thegRNA targeting domain), and by binding of a portion of the gRNA moleculeto the RNA-guided nuclease or other effector molecule (e.g., through atleast the gRNA tracr). In embodiments, a gRNA molecule consists of asingle contiguous polynucleotide molecule, referred to herein as a“single guide RNA” or “sgRNA” and the like. In other embodiments, a gRNAmolecule consists of a plurality, usually two, polynucleotide molecules,which are themselves capable of association, usually throughhybridization, referred to herein as a “dual guide RNA” or “dgRNA,” andthe like. gRNA molecules are described in more detail below, butgenerally include a targeting domain and a tracr. In embodiments thetargeting domain and tracr are disposed on a single polynucleotide. Inother embodiments, the targeting domain and tracr are disposed onseparate polynucleotides.

The term “targeting domain” as the term is used in connection with agRNA, is the portion of the gRNA molecule that recognizes, e.g., iscomplementary to, a target sequence, e.g., a target sequence within thenucleic acid of a cell, e.g., within a gene.

The term “crRNA” as the term is used in connection with a gRNA molecule,is a portion of the gRNA molecule that comprises a targeting domain anda region that interacts with a tracr to form a flagpole region.

The term “target sequence” refers to a sequence of nucleic acidscomplimentary, for example fully complementary, to a gRNA targetingdomain. In embodiments, the target sequence is disposed on genomic DNA.In an embodiment the target sequence is adjacent to (either on the samestrand or on the complementary strand of DNA) a protospacer adjacentmotif (PAM) sequence recognized by a protein having nuclease or othereffector activity, e.g., a PAM sequence recognized by Cas9. Inembodiments, the target sequence is a target sequence within a gene orlocus that affects expression of a globin gene, e.g., that affectsexpression of beta globin or fetal hemoglobin (HbF). In embodiments, thetarget sequence is a target sequence within a nondeletional HPFH region,for example, is within a HBG1 and/or HBG2 promoter region.

The term “flagpole” as used herein in connection with a gRNA molecule,refers to the portion of the gRNA where the crRNA and the tracr bind to,or hybridize to, one another.

The term “tracr” as used herein in connection with a gRNA molecule,refers to the portion of the gRNA that binds to a nuclease or othereffector molecule. In embodiments, the tracr comprises nucleic acidsequence that binds specifically to Cas9. In embodiments, the tracrcomprises nucleic acid sequence that forms part of the flagpole.

The terms “Cas9” or “Cas9 molecule” refer to an enzyme from bacterialType II CRISPR/Cas system responsible for DNA cleavage. Cas9 alsoincludes wild-type protein as well as functional and non-functionalmutants thereof. In embodiments, the Cas9 is a Cas9 of S. pyogenes.

The term “complementary” as used in connection with nucleic acid, refersto the pairing of bases, A with T or U, and G with C. The termcomplementary refers to nucleic acid molecules that are completelycomplementary, that is, form A to T or U pairs and G to C pairs acrossthe entire reference sequence, as well as molecules that are at least80%, 85%, 90%, 95%, 99% complementary.

“Template Nucleic Acid” as used in connection with homology-directedrepair or homologous recombination, refers to nucleic acid to beinserted at the site of modification by the CRISPR system donor sequencefor gene repair (insertion) at site of cutting.

An “indel,” as the term is used herein, refers to a nucleic acidcomprising one or more insertions of nucleotides, one or more deletionsof nucleotides, or a combination of insertions and deletions ofnucleotides, relative to a reference nucleic acid, that results afterbeing exposed to a composition comprising a gRNA molecule, for example aCRISPR system. Indels can be determined by sequencing nucleic acid afterbeing exposed to a composition comprising a gRNA molecule, for example,by NGS. With respect to the site of an indel, an indel is said to be “ator near” a reference site (e.g., a site complementary to a targetingdomain of a gRNA molecule) if it comprises at least one insertion ordeletion within about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide(s) ofthe reference site, or is overlapping with part or all of said referencesite (e.g., comprises at least one insertion or deletion overlappingwith, or within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides of a sitecomplementary to the targeting domain of a gRNA molecule, e.g., a gRNAmolecule described herein). In embodiments, the indel is a largedeletion, for example, comprising more than about 1 kb, more than about2 kb, more than about 3 kb, more than about 4 kb, more than about 5 kb,more than about 6 kb, or more than about 10 kb of nucleic acid. Inembodiments, the 5′ end, the 3′ end, or both the 5′ and 3′ ends of thelarge deletion are disposed at or near a target sequence of a gRNAmolecule described herein. In embodiments, the large deletion comprisesabout 4.9 kb of DNA disposed between a target sequence of a gRNAmolecule, e.g., described herein, disposed within the HBG1 promoterregion and a target sequence of a gRNA molecule, e.g., described herein,disposed within the HBG2 promoter region.

An “indel pattern,” as the term is used herein, refers to a set ofindels that results after exposure to a composition comprising a gRNAmolecule. In an embodiment, the indel pattern consists of the top threeindels, by frequency of appearance. In an embodiment, the indel patternconsists of the top five indels, by frequency of appearance. In anembodiment, the indel pattern consists of the indels which are presentat greater than about 1% frequency relative to all sequencing reads. Inan embodiment, the indel pattern consists of the indels which arepresent at greater than about 5% frequency relative to all sequencingreads. In an embodiment, the indel pattern consists of the indels whichare present at greater than about 10% frequency relative to total numberof indel sequencing reads (i.e., those reads that do not consist of theunmodified reference nucleic acid sequence). In an embodiment, the indelpattern includes of any 3 of the top five most frequently observedindels. The indel pattern may be determined, for example, by methodsdescribed herein, e.g., by sequencing cells of a population of cellswhich were exposed to the gRNA molecule.

An “off-target indel,” as the term is used herein, refers to an indel ator near a site other than the target sequence of the targeting domain ofthe gRNA molecule. Such sites may comprise, for example, 1, 2, 3, 4, 5or more mismatch nucleotides relative to the sequence of the targetingdomain of the gRNA. In exemplary embodiments, such sites are detectedusing targeted sequencing of in silico predicted off-target sites, or byan insertional method known in the art. With respect to the gRNAsdescribed herein, examples of off-target indels are indels formed atsequences outside of the HBG1 and/or HBG2 promoter regions. In exemplaryembodiments the off-target indel is formed in a sequence of a gene,e.g., within a coding sequence of a gene.

The term “a” and “an” refers to one or to more than one (i.e., to atleast one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

The term “about” when referring to a measurable value such as an amount,a temporal duration, and the like, is meant to encompass variations of±20% or in some instances ±10%, or in some instances ±5%, or in someinstances ±1%, or in some instances ±0.1% from the specified value, assuch variations are appropriate to perform the disclosed methods.

The term “antigen” or “Ag” refers to a molecule that provokes an immuneresponse. This immune response may involve either antibody production,or the activation of specific immunologically-competent cells, or both.The skilled artisan will understand that any macromolecule, includingvirtually all proteins or peptides, can serve as an antigen.Furthermore, antigens can be derived from recombinant or genomic DNA. Askilled artisan will understand that any DNA, which comprises anucleotide sequences or a partial nucleotide sequence encoding a proteinthat elicits an immune response therefore encodes an “antigen” as thatterm is used herein. Furthermore, one skilled in the art will understandthat an antigen need not be encoded solely by a full length nucleotidesequence of a gene. It is readily apparent that the present inventionincludes, but is not limited to, the use of partial nucleotide sequencesof more than one gene and that these nucleotide sequences are arrangedin various combinations to encode polypeptides that elicit the desiredimmune response. Moreover, a skilled artisan will understand that anantigen need not be encoded by a “gene” at all. It is readily apparentthat an antigen can be synthesized or can be derived from a biologicalsample, or might be macromolecule besides a polypeptide. Such abiological sample can include, but is not limited to a tissue sample, acell or a fluid with other biological components.

The term “autologous” refers to any material derived from the sameindividual into whom it is later to be re-introduced.

The term “allogeneic” refers to any material derived from a differentanimal of the same species as the individual to whom the material isintroduced. Two or more individuals are said to be allogeneic to oneanother when the genes at one or more loci are not identical. In someaspects, allogeneic material from individuals of the same species may besufficiently unlike genetically to interact antigenically

The term “xenogeneic” refers to a graft derived from an animal of adifferent species.

“Derived from” as that term is used herein, indicates a relationshipbetween a first and a second molecule. It generally refers to structuralsimilarity between the first molecule and a second molecule and does notconnotate or include a process or source limitation on a first moleculethat is derived from a second molecule.

The term “encoding” refers to the inherent property of specificsequences of nucleotides in a polynucleotide, such as a gene, a cDNA, oran mRNA, to serve as templates for synthesis of other polymers andmacromolecules in biological processes having either a defined sequenceof nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence ofamino acids and the biological properties resulting therefrom. Thus, agene, cDNA, or RNA, encodes a protein if transcription and translationof mRNA corresponding to that gene produces the protein in a cell orother biological system. Both the coding strand, the nucleotide sequenceof which is identical to the mRNA sequence and is usually provided insequence listings, and the non-coding strand, used as the template fortranscription of a gene or cDNA, can be referred to as encoding theprotein or other product of that gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or a RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “effective amount” or “therapeutically effective amount” areused interchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result.

The term “endogenous” refers to any material from or produced inside anorganism, cell, tissue or system.

The term “exogenous” refers to any material introduced from or producedoutside an organism, cell, tissue or system.

The term “expression” refers to the transcription and/or translation ofa particular nucleotide sequence driven by a promoter.

The term “transfer vector” refers to a composition of matter whichcomprises an isolated nucleic acid and which can be used to deliver theisolated nucleic acid to the interior of a cell. Numerous vectors areknown in the art including, but not limited to, linear polynucleotides,polynucleotides associated with ionic or amphiphilic compounds,plasmids, and viruses. Thus, the term “transfer vector” includes anautonomously replicating plasmid or a virus. The term should also beconstrued to further include non-plasmid and non-viral compounds whichfacilitate transfer of nucleic acid into cells, such as, for example, apolylysine compound, liposome, and the like. Examples of viral transfervectors include, but are not limited to, adenoviral vectors,adeno-associated virus vectors, retroviral vectors, lentiviral vectors,and the like.

The term “expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, including cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

The term “homologous” or “identity” refers to the subunit sequenceidentity between two polymeric molecules, e.g., between two nucleic acidmolecules, such as, two DNA molecules or two RNA molecules, or betweentwo polypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit; e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous or identical at that position. The homology between twosequences is a direct function of the number of matching or homologouspositions; e.g., if half (e.g., five positions in a polymer ten subunitsin length) of the positions in two sequences are homologous, the twosequences are 50% homologous; if 90% of the positions (e.g., 9 of 10),are matched or homologous, the two sequences are 90% homologous.

The term “isolated” means altered or removed from the natural state. Forexample, a nucleic acid or a peptide naturally present in a livinganimal is not “isolated,” but the same nucleic acid or peptide partiallyor completely separated from the coexisting materials of its naturalstate is “isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

The term “operably linked” or “transcriptional control” refers tofunctional linkage between a regulatory sequence and a heterologousnucleic acid sequence resulting in expression of the latter. Forexample, a first nucleic acid sequence is operably linked with a secondnucleic acid sequence when the first nucleic acid sequence is placed ina functional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.Operably linked DNA sequences can be contiguous with each other and,e.g., where necessary to join two protein coding regions, are in thesame reading frame.

The term “parenteral” administration of an immunogenic compositionincludes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular(i.m.), or intrasternal injection, intratumoral, or infusion techniques.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacids (DNA) or ribonucleic acids (RNA) and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)).

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. A polypeptide includes a natural peptide, arecombinant peptide, or a combination thereof.

The term “promoter” refers to a DNA sequence recognized by the syntheticmachinery of the cell, or introduced synthetic machinery, required toinitiate the specific transcription of a polynucleotide sequence.

The term “promoter/regulatory sequence” refers to a nucleic acidsequence which is required for expression of a gene product operablylinked to the promoter/regulatory sequence. In some instances, thissequence may be the core promoter sequence and in other instances, thissequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

The term “constitutive” promoter refers to a nucleotide sequence which,when operably linked with a polynucleotide which encodes or specifies agene product, causes the gene product to be produced in a cell undermost or all physiological conditions of the cell.

The term “inducible” promoter refers to a nucleotide sequence which,when operably linked with a polynucleotide which encodes or specifies agene product, causes the gene product to be produced in a cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

The term “tissue-specific” promoter refers to a nucleotide sequencewhich, when operably linked with a polynucleotide encodes or specifiedby a gene, causes the gene product to be produced in a cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

As used herein in connection with a messenger RNA (mRNA), a 5′ cap (alsotermed an RNA cap, an RNA 7-methylguanosine cap or an RNA m7G cap) is amodified guanine nucleotide that has been added to the “front” or 5′ endof a eukaryotic messenger RNA shortly after the start of transcription.The 5′ cap consists of a terminal group which is linked to the firsttranscribed nucleotide. Its presence is critical for recognition by theribosome and protection from RNases. Cap addition is coupled totranscription, and occurs co-transcriptionally, such that eachinfluences the other. Shortly after the start of transcription, the 5′end of the mRNA being synthesized is bound by a cap-synthesizing complexassociated with RNA polymerase. This enzymatic complex catalyzes thechemical reactions that are required for mRNA capping. Synthesisproceeds as a multi-step biochemical reaction. The capping moiety can bemodified to modulate functionality of mRNA such as its stability orefficiency of translation.

As used herein, “in vitro transcribed RNA” refers to RNA, preferablymRNA, that has been synthesized in vitro. Generally, the in vitrotranscribed RNA is generated from an in vitro transcription vector. Thein vitro transcription vector comprises a template that is used togenerate the in vitro transcribed RNA.

As used herein, a “poly(A)” is a series of adenosines attached bypolyadenylation to the mRNA. In the preferred embodiment of a constructfor transient expression, the polyA is between 50 and 5000 (SEQ ID NO:190), preferably greater than 64, more preferably greater than 100, mostpreferably greater than 300 or 400. poly(A) sequences can be modifiedchemically or enzymatically to modulate mRNA functionality such aslocalization, stability or efficiency of translation.

As used herein, “polyadenylation” refers to the covalent linkage of apolyadenylyl moiety, or its modified variant, to a messenger RNAmolecule. In eukaryotic organisms, most messenger RNA (mRNA) moleculesare polyadenylated at the 3′ end. The 3′ poly(A) tail is a long sequenceof adenine nucleotides (often several hundred) added to the pre-mRNAthrough the action of an enzyme, polyadenylate polymerase. In highereukaryotes, the poly(A) tail is added onto transcripts that contain aspecific sequence, the polyadenylation signal. The poly(A) tail and theprotein bound to it aid in protecting mRNA from degradation byexonucleases. Polyadenylation is also important for transcriptiontermination, export of the mRNA from the nucleus, and translation.Polyadenylation occurs in the nucleus immediately after transcription ofDNA into RNA, but additionally can also occur later in the cytoplasm.After transcription has been terminated, the mRNA chain is cleavedthrough the action of an endonuclease complex associated with RNApolymerase. The cleavage site is usually characterized by the presenceof the base sequence AAUAAA near the cleavage site. After the mRNA hasbeen cleaved, adenosine residues are added to the free 3′ end at thecleavage site.

As used herein, “transient” refers to expression of a non-integratedtransgene for a period of hours, days or weeks, wherein the period oftime of expression is less than the period of time for expression of thegene if integrated into the genome or contained within a stable plasmidreplicon in the host cell.

As used herein, the terms “treat”, “treatment” and “treating” refer tothe reduction or amelioration of the progression, severity and/orduration of a disorder, e.g., a hemoglobinopathy, or the amelioration ofone or more symptoms (preferably, one or more discernible symptoms) of adisorder, e.g., a hemoglobinopathy, resulting from the administration ofone or more therapies (e.g., one or more therapeutic agents such as agRNA molecule, CRISPR system, or modified cell of the invention). Inspecific embodiments, the terms “treat”, “treatment” and “treating”refer to the amelioration of at least one measurable physical parameterof a hemoglobinopathy disorder, not discernible by the patient. In otherembodiments the terms “treat”, “treatment” and “treating” refer to theinhibition of the progression of a disorder, either physically by, e.g.,stabilization of a discernible symptom, physiologically by, e.g.,stabilization of a physical parameter, or both. In other embodiments theterms “treat”, “treatment” and “treating” refer to the reduction orstabilization of a symptom of a hemoglobinopathy, e.g., sickle celldisease or beta-thalassemia.

The term “signal transduction pathway” refers to the biochemicalrelationship between a variety of signal transduction molecules thatplay a role in the transmission of a signal from one portion of a cellto another portion of a cell. The phrase “cell surface receptor”includes molecules and complexes of molecules capable of receiving asignal and transmitting signal across the membrane of a cell.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals, human).

The term, a “substantially purified” cell refers to a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some aspects, thecells are cultured in vitro. In other aspects, the cells are notcultured in vitro.

The term “therapeutic” as used herein means a treatment. A therapeuticeffect is obtained by reduction, suppression, remission, or eradicationof a disease state.

The term “prophylaxis” as used herein means the prevention of orprotective treatment for a disease or disease state.

The term “transfected” or “transformed” or “transduced” refers to aprocess by which exogenous nucleic acid and/or protein is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid and/or protein. The cell includesthe primary subject cell and its progeny.

The term “specifically binds,” refers to a molecule recognizing andbinding with a binding partner (e.g., a protein or nucleic acid) presentin a sample, but which molecule does not substantially recognize or bindother molecules in the sample.

The term “bioequivalent” refers to an amount of an agent other than thereference compound, required to produce an effect equivalent to theeffect produced by the reference dose or reference amount of thereference compound.

“Refractory” as used herein refers to a disease, e.g., ahemoglobinopathy, that does not respond to a treatment. In embodiments,a refractory hemoglobinopathy can be resistant to a treatment before orat the beginning of the treatment. In other embodiments, the refractoryhemoglobinopathy can become resistant during a treatment. A refractoryhemoglobinopathy is also called a resistant hemoglobinopathy.

“Relapsed” as used herein refers to the return of a disease (e.g.,hemoglobinopathy) or the signs and symptoms of a disease such as ahemoglobinopathy after a period of improvement, e.g., after priortreatment of a therapy, e.g., hemoglobinopathy therapy.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Asanother example, a range such as 95-99% identity, includes somethingwith 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This appliesregardless of the breadth of the range.

The term “BCL11a” refers to B-cell lymphoma/leukemia 11A, a RNApolymerase II core promoter proximal region sequence-specific DNAbinding protein, and the gene encoding said protein, together with allintrons and exons. This gene encodes a C2H2 type zinc-finger protein.BCL11A has been found to play a role in the suppression of fetalhemoglobin production. BCL11a is also known as B-Cell CLL/Lymphoma 11A(Zinc Finger Protein), CTIP1, EVI9, Ecotropic Viral Integration Site 9Protein Homolog, COUP-TF-Interacting Protein 1, Zinc Finger Protein 856,KIAA1809, BCL-11A, ZNF856, EVI-9, and B-Cell CLL/Lymphoma 11A. The termencompasses all isoforms and splice variants of BLC11a. The human geneencoding BCL11a is mapped to chromosomal location 2p16.1 (by Ensembl).The human and murine amino acid and nucleic acid sequences can be foundin a public database, such as GenBank, UniProt and Swiss-Prot., and thegenomic sequence of human BCL11a can be found in GenBank atNC_000002.12. The BCL11a gene refers to this genomic location, includingall introns and exons. There are multiple known isotypes of BCL11a.

The sequence of mRNA encoding isoform 1 of human BCL11a can be found atNM_022893.

The peptide sequence of isoform 1 of human BCL11a is:

        10         20         30         40 MSRRKQGKPQ HLSKREFSPEPLEAILTDDE PDHGPLGAPE         50         60         70         80GDHDLLTCGQ CQMNFPLGDI LIFIEHKRKQ CNGSLCLEKA        90        100        110        120 VDKPPSPSPI EMKKASNPVEVGIQVTPEDD DCLSTSSRGI        130        140        150        160CPKQEHIADK LLHWRGLSSP RSAHGALIPT PGMSAEYAPQ       170        180        190        200 GICKDEPSSY TCTTCKQPFTSAWFLLQHAQ NTHGLRIYLE        210        220        230        240SEHGSPLTPR VGIPSGLGAE CPSQPPLHGI HIADNNPFNL       250        260        270        280 LRIPGSVSRE ASGLAEGRFPPTPPLFSPPP RHHLDPHRIE        290        300        310        320RLGAEEMALA THHPSAFDRV LRLNPMAMEP PAMDFSRRLR       330        340        350        360 ELAGNTSSPP LSPGRPSPMQRLLQPFQPGS KPPFLATPPL        370        380        390        400PPLQSAPPPS QPPVKSKSCE FCGKTFKFQS NLVVHRRSHT       410        420        430        440 GEKPYKCNLC DHACTQASKLKRHMKTHMHK SSPMTVKSDD        450        460        470        480GLSTASSPEP GTSDLVGSAS SALKSVVAKF KSENDPNLIP       490        500        510        520 ENGDEEEEED DEEEEEEEEEEEEELTESER VDYGFGLSLE        530        540        550        560AARHHENSSR GAVVGVGDES RALPDVMQGM VLSSMQHFSE       570        580        590        600 AFHQVLGEKH KRGHLAEAEGHRDTCDEDSV AGESDRIDDG        610        620        630        640TVNGRGCSPG ESASGGLSKK LLLGSPSSLS PFSKRIKLEK       650        660        670        680 EFDLPPAAMP NTENVYSQWLAGYAASRQLK DPFLSFGDSR        690        700        710        720QSPFASSSEH SSENGSLRFS TPPGELDGGI SGRSGTGSGG       730        740        750        760 STPHISGPGP GRPSSKEGRRSDTCEYCGKV FKNCSNLTVH        770        780        790        800RRSHTGERPY KCELCNYACA QSSKLTRHMK THGQVGKDVY       810        820        830 KCEICKMPFS VYSTLEKHMK KWHSDRVLNN DIKTE

SEQ ID NO: 245 (Identifier Q9H165-1; and NM_022893.3; and accessionADL14508.1).

The sequences of other BCL11a protein isoforms are provided at:

Isoform 2: Q9H165-2

Isoform 3: Q9H165-3

Isoform 4: Q9H165-4

Isoform 5: Q9H165-5

Isoform 6: Q9H165-6

As used herein, a human BCL11a protein also encompasses proteins thathave over its full length at least about 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity with BCL11a isoform 1-6, wherein such proteins still have atleast one of the functions of BCL11a.

The term “globin locus” as used herein refers to the region of humanchromosome 11 comprising genes for embryonic (ε), fetal (G(γ) and A(γ)),adult globin genes (γ and β), locus control regions and DNase Ihypersensitivity sites.

The term “complementary” as used in connection with nucleic acid, refersto the pairing of bases, A with T or U, and G with C. The termcomplementary refers to nucleic acid molecules that are completelycomplementary, that is, form A to T or U pairs and G to C pairs acrossthe entire reference sequence, as well as molecules that are at least80%, 85%, 90%, 95%, 99% complementary.

The term “Nondeletional HPFH” refers to a mutation that does notcomprise an insertion or deletion of one or more nucleotides, whichresults in hereditary persistence of fetal hemoglobin, and ischaracterized in increased fetal hemoglobin in adult red blood cells. Inexemplary embodiments, the nondeletional HPFH is a mutation described inNathan and Oski's Hematology and Oncology of Infancy and Childhood,8^(th) Ed., 2015, Orkin S H, Fisher D E, Look T, Lux S E, Ginsburg D,Nathan D G, Eds., Elsevier Saunders, the entire contents of which isincorporated herein by reference, for example the nondeltional HPFHmutations described at Table 21-5. The term “Nondeletional HPFH region”refers to a genomic site which comprises or is near a nondeletionalHPFH. In exemplary embodiments, the nondeletional HPFH region is thenucleic acid sequence of the HBG1 promoter region (Chr11:5,249,833 toChr11:5,250,237, hg38; − strand), the nucleic acid sequence of the HBG2promoter region (Chr11:5,254,738 to Chr11:5,255,164, hg38; − strand), orcombinations thereof. In exemplary embodiments, the nondeletional HPFHregion includes one or more of the nondeletional HPFH described inNathan and Oski's Hematology and Oncology of Infancy and Childhood,8^(th) Ed., 2015, Orkin S H, Fisher D E, Look T, Lux S E, Ginsburg D,Nathan D G, Eds., Elsevier Saunders (e.g., described in Table 21-5therein). In exemplary embodiments, the nondeletional HPFH region is thenucleic acid sequence at chr11:5,250,094-5,250,237, − strand, hg38; orthe nucleic acid sequence at chr11:5,255,022-5,255,164, − strand, hg38;or the nucleic acid sequence at chr11:5,249,833-5,249,927, − strand,hg38; or the nucleic acid sequence at chr11:5,254,738-5,254,851, −strand, hg38; or the nucleic acid sequence at chr11:5,250,139-5,250,237,− strand, hg38; or combinations thereof.

“BCL11a enhancer” as the term is used herein, refers to nucleic acidsequence which affects, e.g., enhances, expression or function ofBCL11a. See e.g., Bauer et al., Science, vol. 342, 2013, pp. 253-257.The BCL11a enhancer may be, for example, operative only in certain celltypes, for example, cells of the erythroid lineage. One example of aBCL11a enhancer is the nucleic acid sequence between exon 2 and exon 3of the BCL11a gene gene (e.g., the nucleic acid at or corresponding topositions +55: Chr2:60497676-60498941; +58: Chr2:60494251-60495546; +62:Chr2:60490409-60491734 as recorded in hg38). In an embodiment, theBCL11a Enhancer is the +62 region of the nucleic acid sequence betweenexon 2 and exon 3 of the BCL11a gene. In an embodiment, the BCL11aEnhancer is the +58 region of the nucleic acid sequence between exon 2and exon 3 of the BCL11a gene. In an embodiment, the BCL11a Enhancer isthe +55 region of the nucleic acid sequence between exon 2 and exon 3 ofthe BCL11a gene.

The terms “hematopoietic stem and progenitor cell” or “HSPC” are usedinterchangeably, and refer to a population of cells comprising bothhematopoietic stem cells (“HSCs”) and hematopoietic progenitor cells(“HPCs”). Such cells are characterized, for example, as CD34+. Inexemplary embodiments, HSPCs are isolated from bone marrow. In otherexemplary embodiments, HSPCs are isolated from peripheral blood. Inother exemplary embodiments, HSPCs are isolated from umbilical cordblood. In an embodiment, HSPCs are characterized asCD34+/CD38−/CD90+/CD45RA−. In embodiments, the HSPCs are characterizedas CD34+/CD90+/CD49f+ cells. In embodiments, the HSPCs are characterizedas CD34+ cells. In embodiments, the HSPC s are characterized asCD34+/CD90+ cells. In embodiments, the HSPCs are characterized asCD34+/CD90+/CD45RA− cells.

“Stem cell expander” as used herein refers to a compound which causescells, e.g., HSPCs, HSCs and/or HPCs to proliferate, e.g., increase innumber, at a faster rate relative to the same cell types absent saidagent. In one exemplary aspect, the stem cell expander is an inhibitorof the aryl hydrocarbon receptor pathway. Additional examples of stemcell expanders are provided below. In embodiments, the proliferation,e.g., increase in number, is accomplished ex vivo.

“Engraftment” or “engraft” refers to the incorporation of a cell ortissue, e.g., a population of HSPCs, into the body of a recipient, e.g.,a mammal or human subject. In one example, engraftment includes thegrowth, expansion and/or differention of the engrafted cells in therecipient. In an example, engraftment of HSPCs includes thedifferentiation and growth of said HSPCs into erythroid cells within thebody of the recipient.

The term “Hematopoietic progenitor cells” (HPCs) as used herein refersto primitive hematopoietic cells that have a limited capacity forself-renewal and the potential for multilineage differentiation (e.g.,myeloid, lymphoid), mono-lineage differentiation (e.g., myeloid orlymphoid) or cell-type restricted differentiation (e.g., erythroidprogenitor) depending on placement within the hematopoietic hierarchy(Doulatov et al., Cell Stem Cell 2012).

“Hematopoietic stem cells” (HSCs) as used herein refer to immature bloodcells having the capacity to self-renew and to differentiate into moremature blood cells comprising granulocytes (e.g., promyelocytes,neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes,erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producingmegakaryocytes, platelets), and monocytes (e.g., monocytes,macrophages). HSCs are interchangeably described as stem cellsthroughout the specification. It is known in the art that such cells mayor may not include CD34+ cells. CD34+ cells are immature cells thatexpress the CD34 cell surface marker. CD34+ cells are believed toinclude a subpopulation of cells with the stem cell properties definedabove. It is well known in the art that HSCs are multipotent cells thatcan give rise to primitive progenitor cells (e.g., multipotentprogenitor cells) and/or progenitor cells committed to specifichematopoietic lineages (e.g., lymphoid progenitor cells). The stem cellscommitted to specific hematopoietic lineages may be of T cell lineage, Bcell lineage, dendritic cell lineage, Langerhans cell lineage and/orlymphoid tissue-specific macrophage cell lineage. In addition, HSCs alsorefer to long term HSC (LT-HSC) and short term HSC (ST-HSC). ST-HSCs aremore active and more proliferative than LT-HSCs. However, LT-HSC haveunlimited self renewal (i.e., they survive throughout adulthood),whereas ST-HSC have limited self renewal (i.e., they survive for only alimited period of time). Any of these HSCs can be used in any of themethods described herein. Optionally, ST-HSCs are useful because theyare highly proliferative and thus, quickly increase the number of HSCsand their progeny. Hematopoietic stem cells are optionally obtained fromblood products. A blood product includes a product obtained from thebody or an organ of the body containing cells of hematopoietic origin.Such sources include un-fractionated bone marrow, umbilical cord,peripheral blood (e.g., mobilized peripheral blood, e.g., mobilized witha mobilization agent such as G-CSF or Plerixafor® (AMD3100), or acombination of G-CSF and Plerixafor® (AMD3100)), liver, thymus, lymphand spleen. All of the aforementioned crude or un-fractionated bloodproducts can be enriched for cells having hematopoietic stem cellcharacteristics in ways known to those of skill in the art. In anembodiment, HSCs are characterized as CD34+/CD38−/CD90+/CD45RA−. Inembodiments, the HSCs are characterized as CD34+/CD90+/CD49f+ cells. Inembodiments, the HSCs are characterized as CD34+ cells. In embodiments,the HSCs are characterized as CD34+/CD90+ cells. In embodiments, theHSCs are characterized as CD34+/CD90+/CD45RA− cells.

“Expansion” or “Expand” in the context of cells refers to an increase inthe number of a characteristic cell type, or cell types, from an initialcell population of cells, which may or may not be identical. The initialcells used for expansion may not be the same as the cells generated fromexpansion.

“Cell population” refers to eukaryotic mammalian, preferably human,cells isolated from biological sources, for example, blood product ortissues and derived from more than one cell.

“Enriched” when used in the context of cell population refers to a cellpopulation selected based on the presence of one or more markers, forexample, CD34+.

The term “CD34+ cells” refers to cells that express at their surfaceCD34 marker. CD34+ cells can be detected and counted using for exampleflow cytometry and fluorescently labeled anti-CD34 antibodies.

“Enriched in CD34+ cells” means that a cell population has been selectedbased on the presence of CD34 marker. Accordingly, the percentage ofCD34+ cells in the cell population after selection method is higher thanthe percentage of CD34+ cells in the initial cell population beforeselecting step based on CD34 markers. For example, CD34+ cells mayrepresent at least 50%, 60%, 70%, 80% or at least 90% of the cells in acell population enriched in CD34+ cells.

The terms “F cell” and “F-cell” refer to cells, usually erythrocytes(e.g., red blood cells) which contain and/or produce (e.g., express)fetal hemoglobin. For example, an F-cell is a cell that contains orproduces detectible levels of fetal hemoglobin. For example, an F-cellis a cell that contains or produces at least 5 picograms of fetalhemoglobin. In another example, an F-cell is a cell that contains orproduces at least 6 picograms of fetal hemoglobin. In another example,an F-cell is a cell that contains or produces at least 7 picograms offetal hemoglobin. In another example, an F-cell is a cell that containsor produces at least 8 picograms of fetal hemoglobin. In anotherexample, an F-cell is a cell that contains or produces at least 9picograms of fetal hemoglobin. In another example, an F-cell is a cellthat contains or produces at least 10 picograms of fetal hemoglobin.Levels of fetal hemoglobin may be measured using an assay describedherein or by other method known in the art, for example, flow cytometryusing an anti-fetal hemoglobin detection reagent, high performanceliquid chromatography, mass spectrometry, or enzyme-linkedimmunoabsorbent assay.

Unless otherwise stated, all genome or chromosome coordinates areaccording to hg38.

DETAILED DESCRIPTION

The gRNA molecules, compositions and methods described herein relate togenome editing in eukaryotic cells using the CRISPR/Cas9 system. Inparticular, the gRNA molecules, compositions and methods describedherein relate to regulation of globin levels and are useful, forexample, in regulating expression and production of globin genes andprotein. The gRNA molecules, compositions and methods can be useful inthe treatment of hemoglobinopathies.

I. gRNA Molecules

A gRNA molecule may have a number of domains, as described more fullybelow, however, a gRNA molecule typically comprises at least a crRNAdomain (comprising a targeting domain) and a tracr. The gRNA moleculesof the invention, used as a component of a CRISPR system, are useful formodifying (e.g., modifying the sequence) DNA at or near a target site.Such modifications include deletions and or insertions that result in,for example, reduced or eliminated expression of a functional product ofthe gene comprising the target site. These uses, and additional uses,are described more fully below.

In an embodiment, a unimolecular, or sgRNA comprises, preferably from 5′to 3′: a crRNA (which contains a targeting domain complementary to atarget sequence and a region that forms part of a flagpole (i.e., acrRNA flagpole region)); a loop; and a tracr (which contains a domaincomplementary to the crRNA flagpole region, and a domain whichadditionally binds a nuclease or other effector molecule, e.g., a Casmolecule, e.g., a Cas9 molecule), and may take the following format(from 5′ to 3′):

[targeting domain]—[crRNA flagpole region]—[optional first flagpoleextension]—[loop]—[optional first tracr extension]—[tracr flagpoleregion]—[tracr nuclease binding domain].

In embodiments, the tracr nuclease binding domain binds to a Casprotein, e.g., a Cas9 protein.

In an embodiment, a bimolecular, or dgRNA comprises two polynucleotides;the first, preferably from 5′ to 3′: a crRNA (which contains a targetingdomain complementary to a target sequence and a region that forms partof a flagpole; and the second, preferrably from 5′ to 3′: a tracr (whichcontains a domain complementary to the crRNA flagpole region, and adomain which additionally binds a nuclease or other effector molecule,e.g., a Cas molecule, e.g., Cas9 molecule), and may take the followingformat (from 5′ to 3′):

Polynucleotide 1 (crRNA): [targeting domain]—[crRNA flagpoleregion]—[optional first flagpole extension]—[optional second flagpoleextension]

Polynucleotide 2 (tracr): [optional first tracr extension]—[tracrflagpole region]—[tracr nuclease binding domain]

In embodiments, the tracr nuclease binding domain binds to a Casprotein, e.g., a Cas9 protein.

In some aspects, the targeting domain comprises or consists of atargeting domain sequence described herein, e.g., a targeting domaindescribed in Table 1, or a targeting domain comprising or consisting of17, 18, 19, or 20 (preferably 20) consecutive nucleotides of a targetingdomain sequence described in Table 1.

In some aspects, the flagpole, e.g., the crRNA flagpole region,comprises, from 5′ to 3′:

(SEQ ID NO: 182) GUUUUAGAGCUA.

In some aspects, the flagpole, e.g., the crRNA flagpole region,comprises, from 5′ to 3′:

(SEQ ID NO: 183) GUUUAAGAGCUA.

In some aspects the loop comprises, from 5′ to 3′: GAAA (SEQ ID NO:186).

In some aspects the tracr comprises, from 5′ to 3′:UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG UGC (SEQ IDNO: 187) and is preferably used in a gRNA molecule comprising SEQ ID NO182.

In some aspects the tracr comprises, from 5′ to 3′:UAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG UGC (SEQ IDNO: 188) and is preferably used in a gRNA molecule comprising SEQ ID NO183.

In some aspects, the gRNA may also comprise, at the 3′ end, additional Unucleic acids. For example the gRNA may comprise an additional 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 U nucleic acids (SEQ ID NO: 249) at the 3′ end.In an embodiment, the gRNA comprises an additional 4 U nucleic acids atthe 3′ end. In the case of dgRNA, one or more of the polynucleotides ofthe dgRNA (e.g., the polynucleotide comprising the targeting domain andthe polynucleotide comprising the tracr) may comprise, at the 3′ end,additional U nucleic acids. For example, the case of dgRNA, one or moreof the polynucleotides of the dgRNA (e.g., the polynucleotide comprisingthe targeting domain and the polynucleotide comprising the tracr) maycomprise an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 U nucleic acids(SEQ ID NO: 249) at the 3′ end. In an embodiment, in the case of dgRNA,one or more of the polynucleotides of the dgRNA (e.g., thepolynucleotide comprising the targeting domain and the polynucleotidecomprising the tracr) comprises an additional 4 U nucleic acids at the3′ end. In an embodiment of a dgRNA, only the polynucleotide comprisingthe tracr comprises the additional U nucleic acid(s), e.g., 4 U nucleicacids. In an embodiment of a dgRNA, only the polynucleotide comprisingthe targeting domain comprises the additional U nucleic acid(s). In anembodiment of a dgRNA, both the polynucleotide comprising the targetingdomain and the polynucleotide comprising the tracr comprise theadditional U nucleic acids, e.g., 4 U nucleic acids.

In some aspects, the gRNA may also comprise, at the 3′ end, additional Anucleic acids. For example the gRNA may comprise an additional 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 A nucleic acids (SEQ ID NO: 250) at the 3′ end.In an embodiment, the gRNA comprises an additional 4 A nucleic acids atthe 3′ end. In the case of dgRNA, one or more of the polynucleotides ofthe dgRNA (e.g., the polynucleotide comprising the targeting domain andthe polynucleotide comprising the tracr) may comprise, at the 3′ end,additional A nucleic acids. For example, the case of dgRNA, one or moreof the polynucleotides of the dgRNA (e.g., the polynucleotide comprisingthe targeting domain and the polynucleotide comprising the tracr) maycomprise an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 A nucleic acids(SEQ ID NO: 250) at the 3′ end. In an embodiment, in the case of dgRNA,one or more of the polynucleotides of the dgRNA (e.g., thepolynucleotide comprising the targeting domain and the polynucleotidecomprising the tracr) comprises an additional 4 A nucleic acids at the3′ end. In an embodiment of a dgRNA, only the polynucleotide comprisingthe tracr comprises the additional A nucleic acid(s), e.g., 4 A nucleicacids. In an embodiment of a dgRNA, only the polynucleotide comprisingthe targeting domain comprises the additional A nucleic acid(s). In anembodiment of a dgRNA, both the polynucleotide comprising the targetingdomain and the polynucleotide comprising the tracr comprise theadditional U nucleic acids, e.g., 4 A nucleic acids.

In embodiments, one or more of the polynucleotides of the gRNA moleculemay comprise a cap at the 5′ end.

In an embodiment, a unimolecular, or sgRNA comprises, preferably from 5′to 3′: a crRNA (which contains a targeting domain complementary to atarget sequence; a crRNA flagpole region; first flagpole extension; aloop; a first tracr extension (which contains a domain complementary toat least a portion of the first flagpole extension); and a tracr (whichcontains a domain complementary to the crRNA flagpole region, and adomain which additionally binds a Cas9 molecule). In some aspects, thetargeting domain comprises a targeting domain sequence described herein,e.g., a targeting domain described in Table 1, or a targeting domaincomprising or consisting of 17, 18, 19, or 20 (preferably 20)consecutive nucleotides of a targeting domain sequence described inTable 1, for example the 3′ 17, 18, 19, or 20 (preferably 20)consecutive nucleotides of a targeting domain sequence described inTable 1.

In aspects comprising a first flagpole extension and/or a first tracrextension, the flagpole, loop and tracr sequences may be as describedabove. In general any first flagpole extension and first tracr extensionmay be employed, provided that they are complementary. In embodiments,the first flagpole extension and first tracr extension consist of 3, 4,5, 6, 7, 8, 9, 10 or more complementary nucleotides.

In some aspects, the first flagpole extension comprises, from 5′ to 3′:UGCUG (SEQ ID NO: 184). In some aspects, the first flagpole extensionconsists of SEQ ID NO: 184.

In some aspects, the first tracr extension comprises, from 5′ to 3′:CAGCA (SEQ ID NO: 189). In some aspects, the first tracr extensionconsists of SEQ ID NO: 189.

In an embodiment, a dgRNA comprises two nucleic acid molecules. In someaspects, the dgRNA comprises a first nucleic acid which contains,preferably from 5′ to 3′: a targeting domain complementary to a targetsequence; a crRNA flagpole region; optionally a first flagpoleextension; and, optionally, a second flagpole extension; and a secondnucleic acid (which may be referred to herein as a tracr), and comprisesat least a domain which binds a Cas molecule, e.g., a Cas9 molecule)comprising preferably from 5′ to 3′: optionally a first tracr extension;and a tracr (which contains a domain complementary to the crRNA flagpoleregion, and a domain which additionally binds a Cas, e.g., Cas9,molecule). The second nucleic acid may additionally comprise, at the 3′end (e.g., 3′ to the tracr) additional U nucleic acids. For example thetracr may comprise an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Unucleic acids (SEQ ID NO: 249) at the 3′ end (e.g., 3′ to the tracr).The second nucleic acid may additionally or alternately comprise, at the3′ end (e.g., 3′ to the tracr) additional A nucleic acids. For examplethe tracr may comprise an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Anucleic acids (SEQ ID NO: 250) at the 3′ end (e.g., 3′ to the tracr). Insome aspects, the targeting domain comprises a targeting domain sequencedescribed herein, e.g., a targeting domain described in Table 1, or atargeting domain comprising or consisting of 17, 18, 19, or 20(preferably 20) consecutive nucleotides of a targeting domain sequencedescribed in Table 1.

In aspects involving a dgRNA, the crRNA flagpole region, optional firstflagpole extension, optional first tracr extension and tracr sequencesmay be as described above.

In some aspects, the optional second flagpole extension comprises, from5′ to 3′: UUUUG (SEQ ID NO: 185).

In embodiments, the 3′ 1, 2, 3, 4, or 5 nucleotides, the 5′ 1, 2, 3, 4,or 5 nucleotides, or both the 3′ and 5′ 1, 2, 3, 4, or 5 nucleotides ofthe gRNA molecule (and in the case of a dgRNA molecule, thepolynucleotide comprising the targeting domain and/or the polynucleotidecomprising the tracr) are modified nucleic acids, as described morefully in section XIII, below.

The Domains are Discussed Briefly Below:

1) The Targeting Domain:

Guidance on the selection of targeting domains can be found, e.g., in FuY el al. NAT BIOTECHNOL 2014 (doi: 10.1038/nbt.2808) and Sternberg S Hel al. NATURE 2014 (doi: 10.1038/nature13011).

The targeting domain comprises a nucleotide sequence that iscomplementary, e.g., at least 80, 85, 90, 95, or 99% complementary,e.g., fully complementary, to the target sequence on the target nucleicacid. The targeting domain is part of an RNA molecule and will thereforecomprise the base uracil (U), while any DNA encoding the gRNA moleculewill comprise the base thymine (T). While not wishing to be bound bytheory, it is believed that the complementarity of the targeting domainwith the target sequence contributes to specificity of the interactionof the gRNA molecule/Cas9 molecule complex with a target nucleic acid.It is understood that in a targeting domain and target sequence pair,the uracil bases in the targeting domain will pair with the adeninebases in the target sequence.

In an embodiment, the targeting domain is 5 to 50, e.g., 10 to 40, e.g.,10 to 30, e.g., 15 to 30, e.g., 15 to 25 nucleotides in length. In anembodiment, the targeting domain is 15, 16, 17, 18, 19, 20, 21, 22, 23,24 or 25 nucleotides in length. In an embodiment, the targeting domainis 16 nucleotides in length. In an embodiment, the targeting domain is17 nucleotides in length. In an embodiment, the targeting domain is 18nucleotides in length. In an embodiment, the targeting domain is 19nucleotides in length. In an embodiment, the targeting domain is 20nucleotides in length. In embodiments, the aforementioned 16, 17, 18,19, or 20 nucleotides comprise the 5′-16, 17, 18, 19, or 20 nucleotidesfrom a targeting domain described in Table 1. In embodiments, theaforementioned 16, 17, 18, 19, or 20 nucleotides comprise the 3′-16, 17,18, 19, or 20 nucleotides from a targeting domain described in Table 1.

Without being bound by theory, it is believed that the 8, 9, 10, 11 or12 nucleic acids of the targeting domain disposed at the 3′ end of thetargeting domain is important for targeting the target sequence, and maythus be referred to as the “core” region of the targeting domain. In anembodiment, the core domain is fully complementary with the targetsequence.

The strand of the target nucleic acid with which the targeting domain iscomplementary is referred to herein as the target sequence. In someaspects, the target sequence is disposed on a chromosome, e.g., is atarget within a gene. In some aspects the target sequence is disposedwithin an exon of a gene. In some aspects the target sequence isdisposed within an intron of a gene. In some aspects, the targetsequence comprises, or is proximal (e.g., within 10, 20, 30, 40, 50,100, 200, 300, 400, 500, or 1000 nucleic acids) to a binding site of aregulatory element, e.g., a promoter or transcription factor bindingsite, of a gene of interest. Some or all of the nucleotides of thedomain can have a modification, e.g., modification found in Section XIIIherein.

2) crRNA Flagpole Region:

The flagpole contains portions from both the crRNA and the tracr. ThecrRNA flagpole region is complementary with a portion of the tracr, andin an embodiment, has sufficient complementarity to a portion of thetracr to form a duplexed region under at least some physiologicalconditions, for example, normal physiological conditions. In anembodiment, the crRNA flagpole region is 5 to 30 nucleotides in length.In an embodiment, the crRNA flagpole region is 5 to 25 nucleotides inlength. The crRNA flagpole region can share homology with, or be derivedfrom, a naturally occurring portion of the repeat sequence from abacterial CRISPR array. In an embodiment, it has at least 50% homologywith a crRNA flagpole region disclosed herein, e.g., an S. pyogenes, orS. thermophilus, crRNA flagpole region.

In an embodiment, the flagpole, e.g., the crRNA flagpole region,comprises SEQ ID NO: 182. In an embodiment, the flagpole, e.g., thecrRNA flagpole region, comprises sequence having at least 50%, 60%, 70%,80%, 85%, 90%, 95% or 99% homology with SEQ ID NO: 182. In anembodiment, the flagpole, e.g., the crRNA flagpole region, comprises atleast 5, 6, 7, 8, 9, 10, or 11 nucleotides of SEQ ID NO: 182. In anembodiment, the flagpole, e.g., the crRNA flagpole region, comprises SEQID NO: 183. In an embodiment, the flagpole comprises sequence having atleast 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% homology with SEQ ID NO:183. In an embodiment, the flagpole, e.g., the crRNA flagpole region,comprises at least 5, 6, 7, 8, 9, 10, or 11 nucleotides of SEQ ID NO:183.

Some or all of the nucleotides of the domain can have a modification,e.g., modification described in Section XIII herein.

3) First Flagpole Extension

When a tracr comprising a first tracr extension is used, the crRNA maycomprise a first flagpole extension. In general any first flagpoleextension and first tracr extension may be employed, provided that theyare complementary. In embodiments, the first flagpole extension andfirst tracr extension consist of 3, 4, 5, 6, 7, 8, 9, 10 or morecomplementary nucleotides.

The first flagpole extension may comprise nucleotides that arecomplementary, e.g., 80%, 85%, 90%, 95% or 99%, e.g., fullycomplementary, with nucleotides of the first tracr extension. In someaspects, the first flagpole extension nucleotides that hybridize withcomplementary nucleotides of the first tracr extension are contiguous.In some aspects, the first flagpole extension nucleotides that hybridizewith complementary nucleotides of the first tracr extension arediscontinuous, e.g., comprises two or more regions of hybridizationseparated by nucleotides that do not base pair with nucleotides of thefirst tracr extension. In some aspects, the first flagpole extensioncomprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or more nucleotides. In some aspects, the first flagpoleextension comprises, from 5′ to 3′: UGCUG (SEQ ID NO: 184). In someaspects, the first flagpole extension consists of SEQ ID NO: 184. Insome aspects the first flagpole extension comprises nucleic acid that isat least 80%, 85%, 90%, 95% or 99% homology to SEQ ID NO: 184.

Some or all of the nucleotides of the first tracr extension can have amodification, e.g., modification found in Section XIII herein.

3) The Loop

A loop serves to link the crRNA flagpole region (or optionally the firstflagpole extension, when present) with the tracr (or optionally thefirst tracr extension, when present) of a sgRNA. The loop can link thecrRNA flagpole region and tracr covalently or non-covalently. In anembodiment, the linkage is covalent. In an embodiment, the loopcovalently couples the crRNA flagpole region and tracr. In anembodiment, the loop covalently couples the first flagpole extension andthe first tracr extension. In an embodiment, the loop is, or comprises,a covalent bond interposed between the crRNA flagpole region and thedomain of the tracr which hybridizes to the crRNA flagpole region.Typically, the loop comprises one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9,or 10 nucleotides.

In dgRNA molecules the two molecules can be associated by virtue of thehybridization between at least a portion of the crRNA (e.g., the crRNAflagpole region) and at least a portion of the tracr (e.g., the domainof the tracr which is complementary to the crRNA flagpole region).

A wide variety of loops are suitable for use in sgRNAs. Loops canconsist of a covalent bond, or be as short as one or a few nucleotides,e.g., 1, 2, 3, 4, or 5 nucleotides in length. In an embodiment, a loopis 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides inlength. In an embodiment, a loop is 2 to 50, 2 to 40, 2 to 30, 2 to 20,2 to 10, or 2 to 5 nucleotides in length. In an embodiment, a loopshares homology with, or is derived from, a naturally occurringsequence. In an embodiment, the loop has at least 50% homology with aloop disclosed herein. In an embodiment, the loop comprises SEQ ID NO:186.

Some or all of the nucleotides of the domain can have a modification,e.g., modification described in Section XIII herein.

4) The Second Flagpole Extension

In an embodiment, a dgRNA can comprise additional sequence, 3′ to thecrRNA flagpole region or, when present, the first flagpole extension,referred to herein as the second flagpole extension. In an embodiment,the second flagpole extension is, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, or 2-4nucleotides in length. In an embodiment, the second flagpole extensionis 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length. In anembodiment, the second flagpole extension comprises SEQ ID NO: 185.

5) The Tracr:

The tracr is the nucleic acid sequence required for nuclease, e.g.,Cas9, binding Without being bound by theory, it is believed that eachCas9 species is associated with a particular tracr sequence. Tracrsequences are utilized in both sgRNA and in dgRNA systems. In anembodiment, the tracr comprises sequence from, or derived from, an S.pyogenes tracr. In some aspects, the tracr has a portion that hybridizesto the flagpole portion of the crRNA, e.g., has sufficientcomplementarity to the crRNA flagpole region to form a duplexed regionunder at least some physiological conditions (sometimes referred toherein as the tracr flagpole region or a tracr domain complementary tothe crRNA flagpole region). In embodiments, the domain of the tracr thathybridizes with the crRNA flagpole region comprises at least 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides thathybridize with complementary nucleotides of the crRNA flagpole region.In some aspects, the tracr nucleotides that hybridize with complementarynucleotides of the crRNA flagpole region are contiguous. In someaspects, the tracr nucleotides that hybridize with complementarynucleotides of the crRNA flagpole region are discontinuous, e.g.,comprises two or more regions of hybridization separated by nucleotidesthat do not base pair with nucleotides of the crRNA flagpole region. Insome aspects, the portion of the tracr that hybridizes to the crRNAflagpole region comprises, from 5′ to 3′: UAGCAAGUUAAAA (SEQ ID NO:191). In some aspects, the portion of the tracr that hybridizes to thecrRNA flagpole region comprises, from 5′ to 3′: UAGCAAGUUUAAA (SEQ IDNO: 192). In embodiments, the sequence that hybridizes with the crRNAflagpole region is disposed on the tracr 5′- to the sequence of thetracr that additionally binds a nuclease, e.g., a Cas molecule, e.g., aCas9 molecule.

The tracr further comprises a domain that additionally binds to anuclease, e.g., a Cas molecule, e.g., a Cas9 molecule. Without beingbound by theory, it is believed that Cas9 from different species bind todifferent tracr sequences. In some aspects, the tracr comprises sequencethat binds to a S. pyogenes Cas9 molecule. In some aspects, the tracrcomprises sequence that binds to a Cas9 molecule disclosed herein. Insome aspects, the domain that additionally binds a Cas9 moleculecomprises, from 5′ to 3′:UAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 193). Insome aspects the domain that additionally binds a Cas9 moleculecomprises, from 5′ to 3′:

(SEQ ID NO: 194) UAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU UUU.

In some embodiments, the tracr comprises SEQ ID NO: 187. In someembodiments, the tracr comprises SEQ ID NO: 188.

Some or all of the nucleotides of the tracr can have a modification,e.g., modification found in Section XIII herein. In embodiments, thegRNA (e.g., the sgRNA or the tracr and/or crRNA of a dgRNA), e.g., anyof the gRNA or gRNA components described above, comprises an invertedabasic residue at the 5′ end, the 3′ end or both the 5′ and 3′ end ofthe gRNA. In embodiments, the gRNA (e.g., the sgRNA or the tracr and/orcrRNA of a dgRNA), e.g., any of the gRNA or gRNA components describedabove, comprises one or more phosphorothioate bonds between residues atthe 5′ end of the polynucleotide, for example, a phosphorthioate bondbetween the first two 5′ residues, between each of the first three 5′residues, between each of the first four 5′ residues, or between each ofthe first five 5′ residues. In embodiments, the gRNA or gRNA componentmay alternatively or additionally comprise one or more phosphorothioatebonds between residues at the 3′ end of the polynucleotide, for example,a phosphorthioate bond between the first two 3′ residues, between eachof the first three 3′ residues, between each of the first four 3′residues, or between each of the first five 3′ residues. In anembodiment, the gRNA (e.g., the sgRNA or the tracr and/or crRNA of adgRNA), e.g., any of the gRNA or gRNA components described above,comprises a phosphorothioate bond between each of the first four 5′residues (e.g., comprises, e.g., consists of, three phosphorothioatebonds at the 5′ end(s)), and a phosphorothioate bond between each of thefirst four 3′ residues (e.g., comprises, e.g., consists of, threephosphorothioate bonds at the 3′ end(s)). In an embodiment, any of thephosphorothioate modifications described above are combined with aninverted abasic residue at the 5′ end, the 3′ end, or both the 5′ and 3′ends of the polynucleotide. In such embodiments, the inverted abasicnucleotide may be linked to the 5′ and/or 3′ nucleotide by a phosphatebond or a phosphorothioate bond. In embodiments, the gRNA (e.g., thesgRNA or the tracr and/or crRNA of a dgRNA), e.g., any of the gRNA orgRNA components described above, comprises one or more nucleotides thatinclude a 2′ O-methyl modification. In embodiments, each of the first 1,2, 3, or more of the 5′ residues comprise a 2′ O-methyl modification. Inembodiments, each of the first 1, 2, 3, or more of the 3′ residuescomprise a 2′ O-methyl modification. In embodiments, the4^(th)-to-terminal, 3^(rd)-to-terminal, and 2^(nd)-to-terminal 3′residues comprise a 2′ O-methyl modification. In embodiments, each ofthe first 1, 2, 3 or more of the 5′ residues comprise a 2′ O-methylmodification, and each of the first 1, 2, 3 or more of the 3′ residuescomprise a 2′ O-methyl modification. In an embodiment, each of the first3 of the 5′ residues comprise a 2′ O-methyl modification, and each ofthe first 3 of the 3′ residues comprise a 2′ O-methyl modification. Inembodiments, each of the first 3 of the 5′ residues comprise a 2′O-methyl modification, and the 4^(th)-to-terminal, 3^(rd)-to-terminal,and 2^(nd)-to-terminal 3′ residues comprise a 2′ O-methyl modification.In embodiments, any of the 2′ O-methyl modifications, e.g., as describedabove, may be combined with one or more phosphorothioate modifications,e.g., as described above, and/or one or more inverted abasicmodifications, e.g., as described above. In an embodiment, the gRNA(e.g., the sgRNA or the tracr and/or crRNA of a dgRNA), e.g., any of thegRNA or gRNA components described above, comprises, e.g., consists of, aphosphorothioate bond between each of the first four 5′ residues (e.g.,comprises, e.g., consists of three phosphorothioate bonds at the 5′ endof the polynucleotide(s)), a phosphorothioate bond between each of thefirst four 3′ residues (e.g., comprises, e.g., consists of threephosphorothioate bonds at the 5′ end of the polynucleotide(s)), a 2′O-methyl modification at each of the first three 5′ residues, and a 2′O-methyl modification at each of the first three 3′ residues. In anembodiment, the gRNA (e.g., the sgRNA or the tracr and/or crRNA of adgRNA), e.g., any of the gRNA or gRNA components described above,comprises, e.g., consists of, a phosphorothioate bond between each ofthe first four 5′ residues (e.g., comprises, e.g., consists of threephosphorothioate bonds at the 5′ end of the polynucleotide(s)), aphosphorothioate bond between each of the first four 3′ residues (e.g.,comprises, e.g., consists of three phosphorothioate bonds at the 5′ endof the polynucleotide(s)), a 2′ O-methyl modification at each of thefirst three 5′ residues, and a 2′ O-methyl modification at each of the4^(th)-to-terminal, 3^(rd)-to-terminal, and 2^(nd)-to-terminal 3′residues.

In an embodiment, the gRNA (e.g., the sgRNA or the tracr and/or crRNA ofa dgRNA), e.g., any of the gRNA or gRNA components described above,comprises, e.g., consists of, a phosphorothioate bond between each ofthe first four 5′ residues (e.g., comprises, e.g., consists of threephosphorothioate bonds at the 5′ end of the polynucleotide(s)), aphosphorothioate bond between each of the first four 3′ residues (e.g.,comprises, e.g., consists of three phosphorothioate bonds at the 5′ endof the polynucleotide(s)), a 2′ O-methyl modification at each of thefirst three 5′ residues, a 2′ O-methyl modification at each of the firstthree 3′ residues, and an additional inverted abasic residue at each ofthe 5′ and 3′ ends.

In an embodiment, the gRNA (e.g., the sgRNA or the tracr and/or crRNA ofa dgRNA), e.g., any of the gRNA or gRNA components described above,comprises, e.g., consists of, a phosphorothioate bond between each ofthe first four 5′ residues (e.g., comprises, e.g., consists of threephosphorothioate bonds at the 5′ end of the polynucleotide(s)), aphosphorothioate bond between each of the first four 3′ residues (e.g.,comprises, e.g., consists of three phosphorothioate bonds at the 5′ endof the polynucleotide(s)), a 2′ O-methyl modification at each of thefirst three 5′ residues, and a 2′ O-methyl modification at each of the4^(th)-to-terminal, 3^(rd)-to-terminal, and 2^(nd)-to-terminal 3′residues, and an additional inverted abasic residue at each of the 5′and 3′ ends.

In an embodiment, the gRNA is a dgRNA and comprises, e.g., consists of:

crRNA:

mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUUAGAGCUAU*mG*mC*mU (SEQ ID NO: 251), wherem indicates a base with 2′O-Methyl modification, * indicates aphosphorothioate bond, and N's indicate the residues of the targetingdomain, e.g., as described herein, (optionally with an inverted abasicresidue at the 5′ and/or 3′ terminus); and tracr:

AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 224) (optionally with an inverted abasicresidue at the 5′ and/or 3′ terminus).

In an embodiment, the gRNA is a dgRNA and comprises, e.g., consists of:

crRNA:

mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUUAGAGCUAU*mG*mC*mU (SEQ ID NO: 251), wherem indicates a base with 2′O-Methyl modification, * indicates aphosphorothioate bond, and N's indicate the residues of the targetingdomain, e.g., as described herein, (optionally with an inverted abasicresidue at the 5′ and/or 3′ terminus); and tracr:

mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU (SEQ ID NO: 246), where m indicates a basewith 2′O-Methyl modification, * indicates a phosphorothioate bond, andN's indicate the residues of the targeting domain, e.g., as describedherein, (optionally with an inverted abasic residue at the 5′ and/or 3′terminus).

In an embodiment, the gRNA is a dgRNA and comprises, e.g., consists of:

crRNA:

mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGUU*mU*mU*mG (SEQ ID NO: 252),where m indicates a base with 2′O-Methyl modification, * indicates aphosphorothioate bond, and N's indicate the residues of the targetingdomain, e.g., as described herein, (optionally with an inverted abasicresidue at the 5′ and/or 3′ terminus); and

tracr:

AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 224) (optionally with an inverted abasicresidue at the 5′ and/or 3′ terminus).

In an embodiment, the gRNA is a dgRNA and comprises, e.g., consists of:

crRNA:

mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGUU*mU*mU*mG (SEQ ID NO: 252),where m indicates a base with 2′O-Methyl modification, * indicates aphosphorothioate bond, and N's indicate the residues of the targetingdomain, e.g., as described herein, (optionally with an inverted abasicresidue at the 5′ and/or 3′ terminus); and tracr:

mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU (SEQ ID NO: 246), where m indicates a basewith 2′O-Methyl modification, and * indicates a phosphorothioate bond(optionally with an inverted abasic residue at the 5′ and/or 3′terminus).

In an embodiment, the gRNA is a dgRNA and comprises, e.g., consists of:

crRNA:

NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 253), where N'sindicate the residues of the targeting domain, e.g., as describedherein, (optionally with an inverted abasic residue at the 5′ and/or 3′terminus); and tracr:

mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU (SEQ ID NO: 246), where m indicates a basewith 2′O-Methyl modification, and * indicates a phosphorothioate bond(optionally with an inverted abasic residue at the 5′ and/or 3′terminus).

In an embodiment, the gRNA is a sgRNA and comprises, e.g., consists of:

NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 254), where mindicates a base with 2′O-Methyl modification, * indicates aphosphorothioate bond, and N's indicate the residues of the targetingdomain, e.g., as described herein, (optionally with an inverted abasicresidue at the 5′ and/or 3′ terminus).

In an embodiment, the gRNA is a sgRNA and comprises, e.g., consists of:

mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU*mU*mU*mU (SEQ ID NO:255), where m indicates a base with 2′O-Methyl modification, * indicatesa phosphorothioate bond, and N's indicate the residues of the targetingdomain, e.g., as described herein, (optionally with an inverted abasicresidue at the 5′ and/or 3′ terminus).

In an embodiment, the gRNA is a sgRNA and comprises, e.g., consists of:

mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmU*mU*mU*U (SEQ ID NO:256), where m indicates a base with 2′O-Methyl modification, * indicatesa phosphorothioate bond, and N's indicate the residues of the targetingdomain, e.g., as described herein, (optionally with an inverted abasicresidue at the 5′ and/or 3′ terminus).

6) First Tracr Extension

Where the gRNA comprises a first flagpole extension, the tracr maycomprise a first tracr extension. The first tracr extension may comprisenucleotides that are complementary, e.g., 80%, 85%, 90%, 95% or 99%,e.g., fully complementary, with nucleotides of the first flagpoleextension. In some aspects, the first tracr extension nucleotides thathybridize with complementary nucleotides of the first flagpole extensionare contiguous. In some aspects, the first tracr extension nucleotidesthat hybridize with complementary nucleotides of the first flagpoleextension are discontinuous, e.g., comprises two or more regions ofhybridization separated by nucleotides that do not base pair withnucleotides of the first flagpole extension. In some aspects, the firsttracr extension comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. In some aspects, thefirst tracr extension comprises SEQ ID NO: 189. In some aspects thefirst tracr extension comprises nucleic acid that is at least 80%, 85%,90%, 95% or 99% homology to SEQ ID NO: 189.

Some or all of the nucleotides of the first tracr extension can have amodification, e.g., modification found in Section XIII herein.

In some embodiments, the sgRNA may comprise, from 5′ to 3′, disposed 3′to the targeting domain.

a) (SEQ ID NO: 195) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; b) (SEQ ID NO: 196)GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; c) (SEQ ID NO: 197)GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; d) (SEQ ID NO: 198)GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC;

e) any of a) to d), above, further comprising, at the 3′ end, at least1, 2, 3, 4, 5, 6 or 7 uracil (U) nucleotides, e.g., 1, 2, 3, 4, 5, 6, or7 uracil (U) nucleotides;

f) any of a) to d), above, further comprising, at the 3′ end, at least1, 2, 3, 4, 5, 6 or 7 adenine (A) nucleotides, e.g., 1, 2, 3, 4, 5, 6,or 7 adenine (A) nucleotides; or

g) any of a) to f), above, further comprising, at the 5′ end (e.g., atthe 5′ terminus, e.g., 5′ to the targeting domain), at least 1, 2, 3, 4,5, 6 or 7 adenine (A) nucleotides, e.g., 1, 2, 3, 4, 5, 6, or 7 adenine(A) nucleotides. In embodiments, any of a) to g) above is disposeddirectly 3′ to the targeting domain.

In an embodiment, a sgRNA of the invention comprises, e.g., consists of,from 5′ to 3′:

[targeting domain] (SEQ ID NO: 231)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU.

In an embodiment, a sgRNA of the invention comprises, e.g., consists of,from 5′ to 3′:

[targeting domain] (SEQ ID NO: 227)GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU.

In some embodiments, the dgRNA may comprise:

A crRNA comprising, from 5′ to 3′, preferrably disposed directly 3′ tothe targeting domain.

a) (SEQ ID NO: 182) GUUUUAGAGCUA; b) (SEQ ID NO: 183) GUUUAAGAGCUA; c)(SEQ ID NO: 199) GUUUUAGAGCUAUGCUG; d) (SEQ ID NO: 200)GUUUAAGAGCUAUGCUG; e) (SEQ ID NO: 201) GUUUUAGAGCUAUGCUGUUUUG; f) (SEQID NO: 202) GUUUAAGAGCUAUGCUGUUUUG; or g) (SEQ ID NO: 226)GUUUUAGAGCUAUGCU:

and a tracr comprising, from 5′ to 3′:

a) (SEQ ID NO: 187) UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; b) (SEQ ID NO: 188)UAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC GAGUCGGUGC; c) (SEQID NO: 203) CAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; d) (SEQ ID NO: 204)CAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG GCACCGAGUCGGUGC; e)(SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU; f) (SEQ ID NO: 225)AACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU; g) (SEQ ID NO: 232)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAG UGGCACCGAGUCGGUGC h)(SEQ ID NO: 227) GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU; i) (SEQ ID NO: 228)AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGG CACCGAGUCGGUGCUUU; j)(SEQ ID NO: 229) GUUGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU;

k) any of a) to j), above, further comprising, at the 3′ end, at least1, 2, 3, 4, 5, 6 or 7 uracil (U) nucleotides, e.g., 1, 2, 3, 4, 5, 6, or7 uracil (U) nucleotides;

l) any of a) to j), above, further comprising, at the 3′ end, at least1, 2, 3, 4, 5, 6 or 7 adenine (A) nucleotides, e.g., 1, 2, 3, 4, 5, 6,or 7 adenine (A) nucleotides; or

m) any of a) to l), above, further comprising, at the 5′ end (e.g., atthe 5′ terminus), at least 1, 2, 3, 4, 5, 6 or 7 adenine (A)nucleotides, e.g., 1, 2, 3, 4, 5, 6, or 7 adenine (A) nucleotides.

In an embodiment, the sequence of k), above comprises the 3′ sequenceUUUUUU, e.g., if a U6 promoter is used for transcription. In anembodiment, the sequence of k), above, comprises the 3′ sequence UUUU,e.g., if an HI promoter is used for transcription. In an embodiment,sequence of k), above, comprises variable numbers of 3′ U's depending,e.g., on the termination signal of the pol-III promoter used. In anembodiment, the sequence of k), above, comprises variable 3′ sequencederived from the DNA template if a T7 promoter is used. In anembodiment, the sequence of k), above, comprises variable 3′ sequencederived from the DNA template, e.g., if in vitro transcription is usedto generate the RNA molecule. In an embodiment, the sequence of k),above, comprises variable 3′ sequence derived from the DNA template,e.g, if a pol-II promoter is used to drive transcription.

In an embodiment, the crRNA comprises, e.g., consists of, a targetingdomain and, disposed 3′ to the targeting domain (e.g., disposed directly3′ to the targeting domain), a sequence comprising, e.g., consisting of,SEQ ID NO: 201, and the tracr comprises, e.g., consists of

(SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In an embodiment, the crRNA comprises, e.g., consists of, a targetingdomain and, disposed 3′ to the targeting domain (e.g., disposed directly3′ to the targeting domain), a sequence comprising, e.g., consisting of,SEQ ID NO: 202, and the tracr comprises, e.g., consists of,

(SEQ ID NO: 225) AACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In an embodiment, the crRNA comprises, e.g., consists of, a targetingdomain and, disposed 3′ to the targeting domain (e.g., disposed directly3′ to the targeting domain), a sequence comprising, e.g., consisting of,GUUUUAGAGCUAUGCU (SEQ ID NO: 226), and the tracr comprises, e.g.,consists of,

(SEQ ID NO: 227) GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU.

In an embodiment, the crRNA comprises, e.g., consists of, a targetingdomain and, disposed 3′ to the targeting domain (e.g., disposed directly3′ to the targeting domain), a sequence comprising, e.g., consisting of,GUUUUAGAGCUAUGCU (SEQ ID NO: 226), and the tracr comprises, e.g.,consists of,

(SEQ ID NO: 228) AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU.

In an embodiment, the crRNA comprises, e.g., consists of, a targetingdomain and, disposed 3′ to the targeting domain (e.g., disposed directly3′ to the targeting domain), a sequence comprising, e.g., consisting of,GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 201), and the tracr comprises, e.g.,consists of,

(SEQ ID NO: 229) GUUGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU.

II. gRNA Targeting Domains Directed to Nondeletional HPFH Regions

Provided in the table below are targeting domains directed tonondeletional HPFH regions, for gRNA molecules of the present invention,and for use in the various aspects of the present invention, forexample, in altering expression of globin genes, for example, a fetalhemoglobin gene or a hemoglobin beta gene.

TABLE 1 gRNA targeting domains directed to nondeletional HPFH regions.SEQ ID NO: s refer to the gRNA targeting domain sequence. Target genomiclocation Targeting Promoter gRNA targeting genomic location (hg38) 2 (ifSEQ Domain ID Region domain sequence (hg38) 1 strand present) strand IDNO: gRNA targeting domains with target sequences only within the HBG1promoter region GCR- HBG1 AGUCCUGGUA chr11: 5250169-5250189 − 1 0001UCCUCUAUGA GCR- HBG1 AAUUAGCAGU chr11: 5250063-5250083 − 2 0002AUCCUCUUGG GCR- HBG1 AGAAUAAAUU chr11: 5250123-5250143 − 3 0003AGAGAAAAAC GCR- HBG1 AAAAAUUAGC chr11: 5250066-5250086 − 4 0004AGUAUCCUCU GCR- HBG1 AAAAUUAGCA chr11: 5250065-5250085 − 5 0005GUAUCCUCUU GCR- HBG1 AAAAACUGGA chr11: 5250109-5250129 − 6 0006AUGACUGAAU GCR- HBG1 CUCCCAUCAU chr11: 5250163-5250183 + 7 0007AGAGGAUACC GCR- HBG1 GGAGAAGGAA chr11: 5250147-5250167 − 8 0008ACUAGCUAAA GCR- HBG1 GUUUCCUUCU chr11: 5250155-5250175 + 9 0009CCCAUCAUAG GCR- HBG1 GGGAGAAGGA chr11: 5250148-5250168 − 10 0010AACUAGCUAA GCR- HBG1 CACUGGAGCU chr11: 5250213-5250233 − 11 0011AGAGACAAGA GCR- HBG1 AGAGACAAGA chr11: 5250203-5250223 − 12 0012AGGUAAAAAA GCR- HBG1 AAAUUAGCAG chr11: 5250064-5250084 − 13 0013UAUCCUCUUG GCR- HBG1 GUCCUGGUAU chr11: 5250168-5250188 − 14 0014CCUCUAUGAU GCR- HBG1 GUAUCCUCUA chr11: 5250162-5250182 − 15 0015UGAUGGGAGA gRNA targeting domains with target sequences only within theHBG2 promoter region GCR- HBG2 AUUAAGCAGC chr11: 5254990-5255010 − 170017 AGUAUCCUCU GCR- HBG2 AGAAUAAAUU chr11: 5255051-5255071 − 22 0022AGAGAAAAAU GCR- HBG2 AGAAGUCCUG chr11: 5255100-5255120 − 29 0029GUAUCUUCUA GCR- HBG2 UUAAGCAGCA chr11: 5254989-5255009 − 32 0032GUAUCCUCUU GCR- HBG2 AAAAAUUGGA chr11: 5255037-5255057 − 34 0034AUGACUGAAU GCR- HBG2 GGGAGAAGAA chr11: 5255076-5255096 − 46 0046AACUAGCUAA GCR- HBG2 GGAGAAGAAA chr11: 5255075-5255095 − 51 0051ACUAGCUAAA GCR- HBG2 CUCCCACCAU chr11: 5255091-5255111 + 52 0052AGAAGAUACC GCR- HBG2 AGUCCUGGUA chr11: 5255097-5255117 − 54 0054UCUUCUAUGG GCR- HBG2 GUCCUGGUAU chr11: 5255096-5255116 − 58 0058CUUCUAUGGU GCR- HBG2 UAAGCAGCAG chr11: 5254988-5255008 − 60 0060UAUCCUCUUG GCR- HBG2 AAGCAGCAGU chr11: 5254987-5255007 − 69 0069AUCCUCUUGG gRNA with targeting domains within the HBG1 and HBG2 promoterregions GCR- HBG1/HBG2 CCUAGCCAGC chr11: 5249895-5249915 + chr11:5254819-5254839 + 16 0016 CGCCGGCCCC GCR- HBG1/HBG2 UAUCCAGUGA chr11:5249910-5249930 − chr11: 5254834-5254854 − 18 0018 GGCCAGGGGC GCR-HBG1/HBG2 CAUUGAGAUA chr11: 5250036-5250056 + chr11: 5254960-5254980 +19 0019 GUGUGGGGAA GCR- HBG1/HBG2 CCAGUGAGGC chr11: 5249907-5249927 −chr11: 5254831-5254851 − 20 0020 CAGGGGCCGG GCR- HBG1/HBG2 GUGGGGAAGGchr11: 5250048-5250068 + chr11: 5254972-5254992 + 21 0021 GGCCCCCAAGGCR- HBG1/HBG2 CCAGGGGCCG chr11: 5249898-5249918 − chr11:5254822-5254842 − 23 0023 GCGGCUGGCU GCR- HBG1/HBG2 UGAGGCCAGG chr11:5249903-5249923 − chr11: 5254827-5254847 − 24 0024 GGCCGGCGGC GCR-HBG1/HBG2 CAGUUCCACA chr11: 5249846-5249866 − chr11: 5254770-5254790 −25 0025 CACUCGCUUC GCR- HBG1/HBG2 CCGCCGGCCCC chr11: 5249904-5249924 +chr11: 5254828-5254848 + 26 0026 UGGCCUCAC GCR- HBG1/HBG2 GUUUGCCUUGchr11: 5249949-5249969 + chr11: 5254873-5254893 + 27 0027 UCAAGGCUAUGCR- HBG1/HBG2 GGCUAGGGAU chr11: 5249882-5249902 − chr11:5254806-5254826 − 28 0028 GAAGAAUAAA GCR- HBG1/HBG2 CAGGGGCCGG chr11:5249897-5249917 − chr11: 5254821-5254841 − 30 0030 CGGCUGGCUA GCR-HBG1/HBG2 ACUGGAUACU chr11: 5249922-5249942 + chr11: 5254846-5254866 +31 0031 CUAAGACUAU GCR- HBG1/HBG2 CCCUGGCUAA chr11: 5249995-5250015 −chr11: 5254919-5254939 − 33 0033 ACUCCACCCA GCR- HBG1/HBG2 UUAGAGUAUCchr11: 5249916-5249936 − chr11: 5254840-5254860 − 35 0035 CAGUGAGGCCGCR- HBG1/HBG2 CCCAUGGGUG chr11: 5249991-5250011 + chr11:5254915-5254935 + 36 0036 GAGUUUAGCC GCR- HBG1/HBG2 AGGCAAGGCU chr11:5249975-5249995 + chr11: 5254899-5254919 + 37 0037 GGCCAACCCA GCR-HBG1/HBG2 UAGAGUAUCC chr11: 5249915-5249935 − chr11: 5254839-5254859 −38 0038 AGUGAGGCCA GCR- HBG1/HBG2 UAUCUGUCUG chr11: 5250012-5250032 −chr11: 5254936-5254956 − 39 0039 AAACGGUCCC GCR- HBG1/HBG2 AUUGAGAUAGchr11: 5250037-5250057 + chr11: 5254961-5254981 + 40 0040 UGUGGGGAAGGCR- HBG1/HBG2 CUUCAUCCCU chr11: 5249888-5249908 + chr11:5254812-5254832 + 41 0041 AGCCAGCCGC GCR- HBG1/HBG2 GCUAUUGGUC chr11:5249964-5249984 + chr11: 5254888-5254908 + 42 0042 AAGGCAAGGC GCR-HBG1/HBG2 AUGCAAAUAU chr11: 5250019-5250039 − chr11: 5254943-5254963 −43 0043 CUGUCUGAAA GCR- HBG1/HBG2 GCAUUGAGAU chr11: 5250035-5250055 +chr11: 5254959-5254979 + 44 0044 AGUGUGGGGA GCR- HBG1/HBG2 UGGUCAAGUUchr11: 5249942-5249962 + chr11: 5254866-5254886 + 45 0045 UGCCUUGUCAGCR- HBG1/HBG2 GGCAAGGCUG chr11: 5249976-5249996 + chr11:5254900-5254920 + 47 0047 GCCAACCCAU GCR- HBG1/HBG2 ACGGCUGACA chr11:5250184-5250204 − chr11: 5255112-5255132 − 48 0048 AAAGAAGUCC GCR-HBG1/HBG2 CGAGUGUGUG chr11: 5249850-5249870 + chr11: 5254774-5254794 +49 0049 GAACUGCUGA GCR- HBG1/HBG2 CCUGGCUAAA chr11: 5249994-5250014 −chr11: 5254918-5254938 − 50 0050 CUCCACCCAU GCR- HBG1/HBG2 CUUGUCAAGGchr11: 5249955-5249975 + chr11: 5254879-5254899 + 53 0053 CUAUUGGUCAGCR- HBG1/HBG2 AUAUUUGCAU chr11: 5250029-5250049 + chr11:5254953-5254973 + 55 0055 UGAGAUAGUG GCR- HBG1/HBG2 GCUAAACUCC chr11:5249990-5250010 − chr11: 5254914-5254934 − 56 0056 ACCCAUGGGU GCR-HBG1/HBG2 ACGUUCCAGA chr11: 5249838-5249858 + chr11: 5254762-5254782 +57 0057 AGCGAGUGUG GCR- HBG1/HBG2 UAUUUGCAUU chr11: 5250030-5250050 +chr11: 5254954-5254974 + 59 0059 GAGAUAGUGU GCR- HBG1/HBG2 GGAAUGACUGchr11: 5250102-5250122 − chr11: 5255030-5255050 − 61 0061 AAUCGGAACAGCR- HBG1/HBG2 CUUGACCAAU chr11: 5249957-5249977 − chr11:5254881-5254901 − 62 0062 AGCCUUGACA GCR- HBG1/HBG2 CAAGGCUAUU chr11:5249960-5249980 + chr11: 5254884-5254904 + 63 0063 GGUCAAGGCA GCR-HBG1/HBG2 AAGGCUGGCC chr11: 5249979-5249999 + chr11: 5254903-5254923 +64 0064 AACCCAUGGG GCR- HBG1/HBG2 ACUCGCUUCU chr11: 5249835-5249855 −chr11: 5254759-5254779 − 65 0065 GGAACGUCUG GCR- HBG1/HBG2 AUUUGCAUUGchr11: 5250031-5250051 + chr11: 5254955-5254975 + 66 0066 AGAUAGUGUGGCR- HBG1/HBG2 ACUGAAUCGG chr11: 5250096-5250116 − chr11:5255024-5255044 − 67 0067 AACAAGGCAA GCR- HBG1/HBG2 CCAUGGGUGG chr11:5249992-5250012 + chr11: 5254916-5254936 + 68 0068 AGUUUAGCCA GCR-HBG1/HBG2 AGAGUAUCCA chr11: 5249914-5249934 − chr11: 5254838-5254858 −70 0070 GUGAGGCCAG GCR- HBG1/HBG2 GAGUGUGUGG chr11: 5249851-5249871 +chr11: 5254775-5254795 + 71 0071 AACUGCUGAA GCR- HBG1/HBG2 UAGUCUUAGAchr11: 5249921-5249941 − chr11: 5254845-5254865 − 72 0072 GUAUCCAGUG

Table 2, below, shows those targeting domains which, when included in agRNA molecule, result in at least a 17% increase in fetal hemoglobin(e.g., in erythroid cells differentiated from modified HSPCs) at 7 daysaccording to the methods described in the Examples. gRNA moleculescomprising any of these targeting domains are collectively referred toherein as Tier 2 gRNA molecules.

TABLE 2 Targeting Domains for Tier 2 gRNA Molecules Targeting Domain IDGCR-0001 GCR-0006 GCR-0008 GCR-0009 GCR-0010 GCR-0011 GCR-0012 GCR-0028GCR-0034 GCR-0045 GCR-0046 GCR-0047 GCR-0048 GCR-0050 GCR-0051 GCR-0053GCR-0054 GCR-0058 GCR-0062 GCR-0063 GCR-0067

Table 3a and Table 3b, below, show those targeting domains which, whenincluded in a gRNA molecule, result in the highest increase in fetalhemoglobin (e.g., in erythroid cells differentiated from modified HSPCs)at 7 days according to the methods described in the Examples. gRNAmolecules comprising these targeting domains are collectively referredto herein as Tier 1 (e.g., Tirr 1a or Tier 1b) gRNA molecules.

TABLE 3a Targeting Domains for Tier 1a gRNA Molecules Targeting DomainID GCR-0006 GCR-0008 GCR-0028 GCR-0034 GCR-0048 GCR-0067

TABLE 3b Targeting Domains for Tier 1b gRNA Molecules Targeting DomainID GCR-0001 GCR-0008 GCR-0009 GCR-0010 GCR-0012 GCR-0054

III. Methods for Designing gRNAs

Methods for designing gRNAs are described herein, including methods forselecting, designing and validating target sequences. Exemplarytargeting domains are also provided herein. Targeting Domains discussedherein can be incorporated into the gRNAs described herein.

Methods for selection and validation of target sequences as well asoff-target analyses are described, e.g., in. Mali el al., 2013 SCIENCE339(6121): 823-826; Hsu et al, 2013 NAT BIOTECHNOL, 31 (9): 827-32; Fuet al, 2014 NAT BIOTECHNOL, doi: 10.1038/nbt.2808. PubMed PMID:24463574; Heigwer et al, 2014 NAT METHODS 11(2): 122-3. doi:10.1038/nmeth.2812. PubMed PMID: 24481216; Bae el al, 2014BIOINFORMATICS PubMed PMID: 24463181; Xiao A el al, 2014 BIOINFORMATICSPubMed PMID: 24389662.

For example, a software tool can be used to optimize the choice of gRNAwithin a user's target sequence, e.g., to minimize total off-targetactivity across the genome. Off target activity may be other thancleavage. For each possible gRNA choice e.g., using S. pyogenes Cas9,the tool can identify all off-target sequences (e.g., preceding eitherNAG or NGG PAMs) across the genome that contain up to certain number(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base-pairs. Thecleavage efficiency at each off-target sequence can be predicted, e.g.,using an experimentally-derived weighting scheme. Each possible gRNA isthen ranked according to its total predicted off-target cleavage; thetop-ranked gRNAs represent those that are likely to have the greateston-target and the least off-target cleavage. Other functions, e.g.,automated reagent design for CRISPR construction, primer design for theon-target Surveyor assay, and primer design for high-throughputdetection and quantification of off-target cleavage via next-gensequencing, can also be included in the tool. Candidate gRNA moleculescan be evaluated by art-known methods or as described herein.

Although software algorithms may be used to generate an initial list ofpotential gRNA molecules, cutting efficiency and specificity will notnecessarily reflect the predicted values, and gRNA molecules typicallyrequire screening in specific cell lines, e.g., primary human celllines, e.g., human HSPCs, e.g., human CD34+ cells, to determine, forexample, cutting efficiency, indel formation, cutting specificity andchange in desired phenotype. These properties may be assayed by themethods described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-0001 (SEQ ID NO: 1, unmodified sequence underlined below), e.g., oneof the gRNA molecules described below, is useful in the CRISPR systems,methods, cells and other aspects and embodiments of the invention,including in aspects involving more than one gRNA molecule, e.g.,described herein:

sgRNA GCR-0001 #1: (SEQ ID NO: 74)AGUCCUGGUAUCCUCUAUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU sgRNA GCR-0001 #2:(SEQ ID NO: 75) mA*mG*mU*CCUGGUAUCCUCUAUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCU*mU*mU*mU sgRNAGCR-0001 #3: (SEQ ID NO: 76)mA*mG*mU*CCUGGUAUCCUCUAUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCmU*mU*mU*U dgRNAGCR-0001 #1: (SEQ ID NO: 77) crRNA:AGUCCUGGUAUCCUCUAUGAGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0001 #2: crRNA: (SEQ ID NO: 78)mA*mG*mU*CCUGGUAUCCUCUAUGAGUUUUAGAGCUAUGCUGUU*mU* mU*mG tracr: (SEQ IDNO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0001 #3: (SEQ ID NO: 78)crRNA: mA*mG*mU*CCUGGUAUCCUCUAUGAGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr:(SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-0006 (SEQ ID NO: 6, unmodified sequence underlined below), e.g., oneof the gRNA molecules described below, is useful in the CRISPR systems,methods, cells and other aspects and embodiments of the invention,including in aspects involving more than one gRNA molecule, e.g.,described herein:

sgRNA GCR-0006 #1: (SEQ ID NO: 79)AAAAACUGGAAUGACUGAAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 80) sgRNAGCR-0006 #2: mA*mA*mA*AACUGGAAUGACUGAAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCU*mU*mU*mU sgRNAGCR-0006 #3: (SEQ ID NO: 81)mA*mA*mA*AACUGGAAUGACUGAAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCmU*mU*mU*U dgRNAGCR-0006 #1: (SEQ ID NO: 82) crRNA:AAAAACUGGAAUGACUGAAUGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0006 #2: (SEQ ID NO: 83) crRNA:mA*mA*mA*AACUGGAAUGACUGAAUGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr: (SEQ IDNO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0006 #3: (SEQ ID NO: 83)crRNA: mA*mA*mA*AACUGGAAUGACUGAAUGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr:(SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-0008 (SEQ ID NO: 8, unmodified sequence underlined below), e.g., oneof the gRNA molecules described below, is useful in the CRISPR systems,methods, cells and other aspects and embodiments of the invention,including in aspects involving more than one gRNA molecule, e.g.,described herein:

sgRNA GCR-0008 #1: (SEQ ID NO: 84)GGAGAAGGAAACUAGCUAAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU sgRNA GCR-0008 #2:(SEQ ID NO: 85) mG*mG*mA*GAAGGAAACUAGCUAAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCU*mU*mU*mU sgRNAGCR-0008 #3: (SEQ ID NO: 86)mG*mG*mA*GAAGGAAACUAGCUAAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCmU*mU*mU*U dgRNAGCR-0008 #1: (SEQ ID NO: 87) crRNA:GGAGAAGGAAACUAGCUAAAGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0008 #2: (SEQ ID NO: 88) crRNA:mG*mG*mA*GAAGGAAACUAGCUAAAGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr: (SEQ IDNO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0008 #3: (SEQ ID NO: 88)crRNA: mG*mG*mA*GAAGGAAACUAGCUAAAGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr:(SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-009 (SEQ ID NO: 9, unmodified sequence underlined below), e.g., oneof the gRNA molecules described below, is useful in the CRISPR systems,methods, cells and other aspects and embodiments of the invention,including in aspects involving more than one gRNA molecule, e.g.,described herein:

sgRNA GCR-0009 #1: (SEQ ID NO: 89)GUUUCCUUCUCCCAUCAUAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU sgRNA GCR-0009 #2:(SEQ ID NO: 90) mG*mU*mU*UCCUUCUCCCAUCAUAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCU*mU*mU*mU sgRNAGCR-0009 #3: (SEQ ID NO: 91)mG*mU*mU*UCCUUCUCCCAUCAUAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCmU*mU*mU*U dgRNAGCR-0009 #1: (SEQ ID NO: 92) crRNA:GUUUCCUUCUCCCAUCAUAGGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0009 #2: (SEQ ID NO: 93) crRNA:mG*mU*mU*UCCUUCUCCCAUCAUAGGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr: (SEQ IDNO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0009 #3: (SEQ ID NO: 93)crRNA: mG*mU*mU*UCCUUCUCCCAUCAUAGGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr:(SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-0010 (SEQ ID NO: 10, unmodified sequence underlined below), e.g.,one of the gRNA molecules described below, is useful in the CRISPRsystems, methods, cells and other aspects and embodiments of theinvention, including in aspects involving more than one gRNA molecule,e.g., described herein:

sgRNA GCR-0010 #1: (SEQ ID NO: 94)GGGAGAAGGAAACUAGCUAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU sgRNA GCR-0010 #2:(SEQ ID NO: 95) mG*mG*mG*AGAAGGAAACUAGCUAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCU*mU*mU*mU sgRNAGCR-0010 #3: (SEQ ID NO: 96)mG*mG*mG*AGAAGGAAACUAGCUAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCmU*mU*mU*U dgRNAGCR-0010 #1: (SEQ ID NO: 97) crRNA:GGGAGAAGGAAACUAGCUAAGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0010 #2: crRNA: (SEQ ID NO: 98)mG*mG*mG*AGAAGGAAACUAGCUAAGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr: (SEQ IDNO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0010 #3: crRNA: (SEQ IDNO: 98) mG*mG*mG*AGAAGGAAACUAGCUAAGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr:(SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-0011 (SEQ ID NO: 11, unmodified sequence underlined below), e.g.,one of the gRNA molecules described below, is useful in the CRISPRsystems, methods, cells and other aspects and embodiments of theinvention, including in aspects involving more than one gRNA molecule,e.g., described herein:

sgRNA GCR-0011 #1: (SEQ ID NO: 99)CACUGGAGCUAGAGACAAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU sgRNA GCR-0011 #2:(SEQ ID NO: 100) mC*mA*mC*UGGAGCUAGAGACAAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCU*mU*mU*mU sgRNAGCR-0011 #3: (SEQ ID NO: 101)mC*mA*mC*UGGAGCUAGAGACAAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCmU*mU*mU*U dgRNAGCR-0011 #1: (SEQ ID NO: 102) crRNA:CACUGGAGCUAGAGACAAGAGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0011 #2: crRNA: (SEQ ID NO: 103)mC*mA*mC*UGGAGCUAGAGACAAGAGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr: (SEQ IDNO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0011 #3: crRNA: (SEQ IDNO: 103) mC*mA*mC*UGGAGCUAGAGACAAGAGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr:(SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-0012 (SEQ ID NO: 12, unmodified sequence underlined below), e.g.,one of the gRNA molecules described below, is useful in the CRISPRsystems, methods, cells and other aspects and embodiments of theinvention, including in aspects involving more than one gRNA molecule,e.g., described herein:

sgRNA GCR-0012 #1: (SEQ ID NO: 104)AGAGACAAGAAGGUAAAAAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU sgRNA GCR-0012 #2:(SEQ ID NO: 105) mA*mG*mA*GACAAGAAGGUAAAAAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCU*mU*mU*mU sgRNAGCR-0012 #3: (SEQ ID NO: 106)mA*mG*mA*GACAAGAAGGUAAAAAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCmU*mU*mU*U dgRNAGCR-0012 #1: (SEQ ID NO: 107) crRNA:AGAGACAAGAAGGUAAAAAAGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0012 #2: crRNA: (SEQ ID NO: 108)mA*mG*mA*GACAAGAAGGUAAAAAAGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr: (SEQ IDNO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0012 #3: crRNA: (SEQ IDNO: 108) mA*mG*mA*GACAAGAAGGUAAAAAAGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr:(SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-0028 (SEQ ID NO: 28, unmodified sequence underlined below), e.g.,one of the gRNA molecules described below, is useful in the CRISPRsystems, methods, cells and other aspects and embodiments of theinvention, including in aspects involving more than one gRNA molecule,e.g., described herein:

sgRNA GCR-0028 #1: (SEQ ID NO: 109)GGCUAGGGAUGAAGAAUAAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU sgRNA GCR-0028 #2:(SEQ ID NO: 110) mG*mG*mC*UAGGGAUGAAGAAUAAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCU*mU*mU*mU sgRNAGCR-0028 #3: (SEQ ID NO: 111)mG*mG*mC*UAGGGAUGAAGAAUAAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCmU*mU*mU*U dgRNAGCR-0028 #1: (SEQ ID NO: 112) crRNA:GGCUAGGGAUGAAGAAUAAAGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0028 #2: crRNA: (SEQ ID NO: 113)mG*mG*mC*UAGGGAUGAAGAAUAAAGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr: (SEQ IDNO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0028 #3: crRNA: (SEQ IDNO: 113) mG*mG*mC*UAGGGAUGAAGAAUAAAGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr:(SEQ ID NO: 224). AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-0034 (SEQ ID NO: 34, unmodified sequence underlined below), e.g.,one of the gRNA molecules described below, is useful in the CRISPRsystems, methods, cells and other aspects and embodiments of theinvention, including in aspects involving more than one gRNA molecule,e.g., described herein:

sgRNA GCR-0034 #1: (SEQ ID NO: 114)AAAAAUUGGAAUGACUGAAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU sgRNA GCR-0034 #2:(SEQ ID NO: 115) mA*mA*mA*AAUUGGAAUGACUGAAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCU*mU*mU*mU sgRNAGCR-0034 #3: (SEQ ID NO: 116)mA*mA*mA*AAUUGGAAUGACUGAAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCmU*mU*mU*U dgRNAGCR-0034 #1: (SEQ ID NO: 117) crRNA:AAAAAUUGGAAUGACUGAAUGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0034 #2: crRNA: (SEQ ID NO: 118)mA*mA*mA*AAUUGGAAUGACUGAAUGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr: (SEQ IDNO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0034 #3: crRNA: (SEQ IDNO: 118) mA*mA*mA*AAUUGGAAUGACUGAAUGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr:(SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-0045 (SEQ ID NO: 45, unmodified sequence underlined below), e.g.,one of the gRNA molecules described below, is useful in the CRISPRsystems, methods, cells and other aspects and embodiments of theinvention, including in aspects involving more than one gRNA molecule,e.g., described herein:

sgRNA GCR-0045 #1: (SEQ ID NO: 119)UGGUCAAGUUUGCCUUGUCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU sgRNA GCR-0045 #2:(SEQ ID NO: 120) mU*mG*mG*UCAAGUUUGCCUUGUCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCU*mU*mU*mU sgRNAGCR-0045 #3: (SEQ ID NO: 121)mU*mG*mG*UCAAGUUUGCCUUGUCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCmU*mU*mU*U dgRNAGCR-0045 #1: (SEQ ID NO: 122) crRNA:UGGUCAAGUUUGCCUUGUCAGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0045 #2: crRNA: (SEQ ID NO: 123)mU*mG*mG*UCAAGUUUGCCUUGUCAGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr: (SEQ IDNO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0045 #3: crRNA: (SEQ IDNO: 123) mU*mG*mG*UCAAGUUUGCCUUGUCAGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr:(SEQ ID NO: 224). AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-0046 (SEQ ID NO: 46, unmodified sequence underlined below), e.g.,one of the gRNA molecules described below, is useful in the CRISPRsystems, methods, cells and other aspects and embodiments of theinvention, including in aspects involving more than one gRNA molecule,e.g., described herein:

sgRNA GCR-0046 #1: (SEQ ID NO: 124)GGGAGAAGAAAACUAGCUAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU sgRNA GCR-0046 #2:(SEQ ID NO: 125) mG*mG*mG*AGAAGAAAACUAGCUAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCU*mU*mU*mU sgRNAGCR-0046 #3: (SEQ ID NO: 126)mG*mG*mG*AGAAGAAAACUAGCUAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCmU*mU*mU*U dgRNAGCR-0046 #1: (SEQ ID NO: 127) crRNA:GGGAGAAGAAAACUAGCUAAGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0046 #2: crRNA: (SEQ ID NO: 128)mG*mG*mG*AGAAGAAAACUAGCUAAGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr: (SEQ IDNO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0046 #3: crRNA: (SEQ IDNO: 128) mG*mG*mG*AGAAGAAAACUAGCUAAGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr:(SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-0047 (SEQ ID NO: 47, unmodified sequence underlined below), e.g.,one of the gRNA molecules described below, is useful in the CRISPRsystems, methods, cells and other aspects and embodiments of theinvention, including in aspects involving more than one gRNA molecule,e.g., described herein:

sgRNA GCR-0047 #1: (SEQ ID NO: 129)GGCAAGGCUGGCCAACCCAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU sgRNA GCR-0047 #2:(SEQ ID NO: 130) mG*mG*mC*AAGGCUGGCCAACCCAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCU*mU*mU*mU sgRNAGCR-0047 #3: (SEQ ID NO: 131)mG*mG*mC*AAGGCUGGCCAACCCAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCmU*mU*mU*U dgRNAGCR-0047 #1: (SEQ ID NO: 132) crRNA:GGCAAGGCUGGCCAACCCAUGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0047 #2: crRNA: (SEQ ID NO: 133)mG*mG*mC*AAGGCUGGCCAACCCAUGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr: (SEQ IDNO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0047 #3: crRNA: (SEQ IDNO: 133) mG*mG*mC*AAGGCUGGCCAACCCAUGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr:(SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-0048 (SEQ ID NO: 48, unmodified sequence underlined below), e.g.,one of the gRNA molecules described below, is useful in the CRISPRsystems, methods, cells and other aspects and embodiments of theinvention, including in aspects involving more than one gRNA molecule,e.g., described herein:

sgRNA GCR-0048 #1: (SEQ ID NO: 134)ACGGCUGACAAAAGAAGUCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU sgRNA GCR-0048 #2:(SEQ ID NO: 135) mA*mC*mG*GCUGACAAAAGAAGUCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCU*mU*mU*mU sgRNAGCR-0048 #3: (SEQ ID NO: 136)mA*mC*mG*GCUGACAAAAGAAGUCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCmU*mU*mU*U dgRNAGCR-0048 #1: (SEQ ID NO: 137) crRNA:ACGGCUGACAAAAGAAGUCCGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0048 #2: crRNA: (SEQ ID NO: 138)mA*mC*mG*GCUGACAAAAGAAGUCCGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr: (SEQ IDNO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0048 #3: (SEQ ID NO: 138)crRNA: mA*mC*mG*GCUGACAAAAGAAGUCCGUUUUAGAGCUAUGCUG UU*mU*mU*mG tracr:(SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-0050 (SEQ ID NO: 50, unmodified sequence underlined below), e.g.,one of the gRNA molecules described below, is useful in the CRISPRsystems, methods, cells and other aspects and embodiments of theinvention, including in aspects involving more than one gRNA molecule,e.g., described herein:

sgRNA GCR-0050 #1: (SEQ ID NO: 139)CCUGGCUAAACUCCACCCAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU sgRNA GCR-0050 #2:(SEQ ID NO: 140) mC*mC*mU*GGCUAAACUCCACCCAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCU*mU*mU*mU sgRNAGCR-0050 #3: (SEQ ID NO: 141)mC*mC*mU*GGCUAAACUCCACCCAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCmU*mU*mU*U dgRNAGCR-0050 #1: (SEQ ID NO: 142) crRNA:CCUGGCUAAACUCCACCCAUGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0050 #2: (SEQ ID NO: 143) crRNA:mC*mC*mU*GGCUAAACUCCACCCAUGUUUUAGAGCUAUGCUG UU*mU*mU*mG tracr: (SEQ IDNO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0050 #3: (SEQ ID NO: 143)crRNA: mC*mC*mU*GGCUAAACUCCACCCAUGUUUUAGAGCUAUGCUG UU*mU*mU*mG tracr:(SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-0051 (SEQ ID NO: 51, unmodified sequence underlined below), e.g.,one of the gRNA molecules described below, is useful in the CRISPRsystems, methods, cells and other aspects and embodiments of theinvention, including in aspects involving more than one gRNA molecule,e.g., described herein:

sgRNA GCR-0051 #1: (SEQ ID NO: 144)GGAGAAGAAAACUAGCUAAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU sgRNA GCR-0051 #2:(SEQ ID NO: 145) mG*mG*mA*GAAGAAAACUAGCUAAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCU*mU*mU*mU sgRNAGCR-0051 #3: (SEQ ID NO: 146)mG*mG*mA*GAAGAAAACUAGCUAAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCmU*mU*mU*U dgRNAGCR-0051 #1: (SEQ ID NO: 147) crRNA:GGAGAAGAAAACUAGCUAAAGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0051 #2: crRNA: (SEQ ID NO: 148)mG*mG*mA*GAAGAAAACUAGCUAAAGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr: (SEQ IDNO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0051 #3: crRNA: (SEQ IDNO: 148) mG*mG*mA*GAAGAAAACUAGCUAAAGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr:(SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-0053 (SEQ ID NO: 53, unmodified sequence underlined below), e.g.,one of the gRNA molecules described below, is useful in the CRISPRsystems, methods, cells and other aspects and embodiments of theinvention, including in aspects involving more than one gRNA molecule,e.g., described herein:

sgRNA GCR-0053 #1: (SEQ ID NO: 149)CUUGUCAAGGCUAUUGGUCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU sgRNA GCR-0053 #2:(SEQ ID NO: 150) mC*mU*mU*GUCAAGGCUAUUGGUCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCU*mU*mU*mU sgRNAGCR-0053 #3: (SEQ ID NO: 151)mC*mU*mU*GUCAAGGCUAUUGGUCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCmU*mU*mU*U dgRNAGCR-0053 #1: (SEQ ID NO: 152) crRNA:CUUGUCAAGGCUAUUGGUCAGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0053 #2: crRNA: (SEQ ID NO: 153)mC*mU*mU*GUCAAGGCUAUUGGUCAGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr: (SEQ IDNO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0053 #3: crRNA: (SEQ IDNO: 153) mC*mU*mU*GUCAAGGCUAUUGGUCAGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr:(SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-0054 (SEQ ID NO: 54, unmodified sequence underlined below), e.g.,one of the gRNA molecules described below, is useful in the CRISPRsystems, methods, cells and other aspects and embodiments of theinvention, including in aspects involving more than one gRNA molecule,e.g., described herein:

sgRNA GCR-0054 #1: (SEQ ID NO: 154)AGUCCUGGUAUCUUCUAUGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU sgRNA GCR-0054 #2:(SEQ ID NO: 155) mA*mG*mU*CCUGGUAUCUUCUAUGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCU*mU*mU*mU sgRNAGCR-0054 #3: (SEQ ID NO: 156)mA*mG*mU*CCUGGUAUCUUCUAUGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCmU*mU*mU*U dgRNAGCR-0054 #1: (SEQ ID NO: 157) crRNA:AGUCCUGGUAUCUUCUAUGGGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0054 #2: crRNA: (SEQ ID NO: 158)mA*mG*mU*CCUGGUAUCUUCUAUGGGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr: (SEQ IDNO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0054 #3: crRNA: (SEQ IDNO: 158) mA*mG*mU*CCUGGUAUCUUCUAUGGGUUUUAGAGCUAUGCUGUU*mU*m U*mG tracr:(SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-0058 (SEQ ID NO: 58, unmodified sequence underlined below), e.g.,one of the gRNA molecules described below, is useful in the CRISPRsystems, methods, cells and other aspects and embodiments of theinvention, including in aspects involving more than one gRNA molecule,e.g., described herein:

sgRNA GCR-0058 #1: (SEQ ID NO: 159)GUCCUGGUAUCUUCUAUGGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU sgRNA GCR-0058 #2:(SEQ ID NO: 160) mG*mU*mC*CUGGUAUCUUCUAUGGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCU*mU*mU*mU sgRNAGCR-0058 #3: (SEQ ID NO: 161)mG*mU*mC*CUGGUAUCUUCUAUGGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA GUCGGUGCmU*mU*mU*U dgRNAGCR-0058 #1: (SEQ ID NO: 162) crRNA:GUCCUGGUAUCUUCUAUGGUGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0058 #2: crRNA: (SEQ ID NO 163)mG*mU*mC*CUGGUAUCUUCUAUGGUGUUUUAGAGCUAUGCUGU U*mU*mU*mG tracr: (SEQ IDNO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0058 #3: crRNA: (SEQ IDNO: 163) mG*mU*mC*CUGGUAUCUUCUAUGGUGUUUUAGAGCUAUGCUGU U*mU*mU*mG tracr:(SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-0062 (SEQ ID NO: 62, unmodified sequence underlined below), e.g.,one of the gRNA molecules described below, is useful in the CRISPRsystems, methods, cells and other aspects and embodiments of theinvention, including in aspects involving more than one gRNA molecule,e.g., described herein:

sgRNA GCR-0062 #1: (SEQ ID NO: 164)CUUGACCAAUAGCCUUGACAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU sgRNA GCR-0062 #2:mC*mU*mU*GACCAAUAGCCUUGACAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCAC CGAGUCGGUGCU*mU*mU*mUsgRNA GCR-0062 #3: (SEQ ID NO: 166)mC*mU*mU*GACCAAUAGCCUUGACAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGCmU*mU*mU*U dgRNAGCR-0062 #1: (SEQ ID NO: 167) crRNA:CUUGACCAAUAGCCUUGACAGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0062 #2: (SEQ ID NO: 168) crRNA:mC*mU*mU*GACCAAUAGCCUUGACAGUUUUAGAGCUAUGCUGU U*mU*mU*mG tracr: (SEQ IDNO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0062 #3: (SEQ ID NO:168) crRNA: mC*mU*mU*GACCAAUAGCCUUGACAGUUUUAGAGCUAUGCU GUU*mU*mU*mGtracr: (SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-0063 (SEQ ID NO: 63, unmodified sequence underlined below), e.g.,one of the gRNA molecules described below, is useful in the CRISPRsystems, methods, cells and other aspects and embodiments of theinvention, including in aspects involving more than one gRNA molecule,e.g., described herein:

sgRNA GCR-0063 #1: (SEQ ID NO: 169)CAAGGCUAUUGGUCAAGGCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU sgRNA GCR-0063 #2:(SEQ ID NO: 170) mC*mA*mA*GGCUAUUGGUCAAGGCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCAC CGAGUCGGUGCU*mU*mU*mUsgRNA GCR-0063 #3: (SEQ ID NO: 171)mC*mA*mA*GGCUAUUGGUCAAGGCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCAC CGAGUCGGUGCmU*mU*mU*UdgRNA GCR-0063 #1: (SEQ ID NO: 172) crRNA:CAAGGCUAUUGGUCAAGGCAGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0063 #2: crRNA: (SEQ ID NO:173) mC*mA*mA*GGCUAUUGGUCAAGGCAGUUUUAGAGCUAUGCUG UU*mU*mU*mG tracr: (SEQID NO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0063 #3: crRNA: (SEQID NO: 173) mC*mA*mA*GGCUAUUGGUCAAGGCAGUUUUAGAGCUAUGCUGU U*mU*mU*mGtracr: (SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

In aspects of the invention, a gRNA comprising the targeting domain ofGCR-0067 (SEQ ID NO: 67, unmodified sequence underlined below), e.g.,one of the gRNA molecules described below, is useful in the CRISPRsystems, methods, cells and other aspects and embodiments of theinvention, including in aspects involving more than one gRNA molecule,e.g., described herein:

sgRNA GCR-0067 #1: (SEQ ID NO: 174)ACUGAAUCGGAACAAGGCAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU sgRNA GCR-0067 #2:(SEQ ID NO: 175) mA*mC*mU*GAAUCGGAACAAGGCAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCAC CGAGUCGGUGCU*mU*mU*mUsgRNA GCR-0067 #3: (SEQ ID NO: 176)mA*mC*mU*GAAUCGGAACAAGGCAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCAC CGAGUCGGUGCmU*mU*mU*UdgRNA GCR-0067 #1: (SEQ ID NO: 177) crRNA:ACUGAAUCGGAACAAGGCAAGUUUUAGAGCUAUGCUGUUUUG tracr: (SEQ ID NO: 224)AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU dgRNA GCR-0067 #2: crRNA: (SEQ ID NO: 178)mA*mC*mU*GAAUCGGAACAAGGCAAGUUUUAGAGCUAUGCUGU U*mU*mU*mG tracr: (SEQ IDNO: 73) mA*mA*mC*AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU*mU*mU*mU dgRNA GCR-0067 #3: crRNA: (SEQID NO: 178) mA*mC*mU*GAAUCGGAACAAGGCAAGUUUUAGAGCUAUGCUGU U*mU*mU*mGtracr: (SEQ ID NO: 224) AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In each of the gRNA molecules described above, a “*” denotes aphosphorothioate bond between the adjacent nucleotides, and “mN” (whereN=A, G, C or U) denotes a 2′-OMe modified nucleotide. In embodiments,any of the gRNA molecules described herein, e.g., described above, iscomplexed with a Cas9 molecule, e.g., as described herein, to form aribonuclear protein complex (RNP). Such RNPs are particularly useful inthe methods, cells, and other aspects and embodiments of the invention,e.g., described herein.

IV. Cas Molecules

Cas9 Molecules

In preferred embodiments, the Cas molecule is a Cas9 molecule. Cas9molecules of a variety of species can be used in the methods andcompositions described herein. While the S. pyogenes Cas9 molecule arethe subject of much of the disclosure herein, Cas9 molecules of, derivedfrom, or based on the Cas9 proteins of other species listed herein canbe used as well. In other words, other Cas9 molecules, e.g., S.thermophilus, Staphylococcus aureus and/or Neisseria meningitidis Cas9molecules, may be used in the systems, methods and compositionsdescribed herein. Additional Cas9 species include: Acidovorax avenae,Actinobacillus pleuropneumoniae, Actinobacillus succinogenes,Actinobacillus suis, Actinomyces sp., Cycliphilus denitrificans,Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillusthuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhiz obiumsp., Brevibacillus latemsporus, Campylobacter coli, Campylobacterjejuni, Campylobacter lad, Candidatus Puniceispirillum, Clostridiucellulolyticum, Clostridium perfringens, Corynebacterium accolens,Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobactersliibae, Eubacterium dolichum, Gamma proteobacterium, Gluconacetobaclerdiazotrophicus, Haemophilus parainfluenzae, Haemophilus sputorum,Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae,Ilyobacler polytropus, Kingella kingae, Lactobacillus crispatus,Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium,Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris,Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens,Neisseria lactamica. Neisseria sp., Neisseria wadsworthii, Nitrosomonassp., Parvibaculum lavamentivorans, Pasteurella multocida,Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonaspalustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp.,Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcussp., Subdoligranulum sp., Tislrella mobilis, Treponema sp., orVerminephrobacter eiseniae.

A Cas9 molecule, as that term is used herein, refers to a molecule thatcan interact with a gRNA molecule (e.g., sequence of a domain of atracr) and, in concert with the gRNA molecule, localize (e.g., target orhome) to a site which comprises a target sequence and PAM sequence.

In an embodiment, the Cas9 molecule is capable of cleaving a targetnucleic acid molecule, which may be referred to herein as an active Cas9molecule. In an embodiment, an active Cas9 molecule, comprises one ormore of the following activities: a nickase activity, i.e., the abilityto cleave a single strand, e.g., the non-complementary strand or thecomplementary strand, of a nucleic acid molecule; a double strandednuclease activity, i.e., the ability to cleave both strands of a doublestranded nucleic acid and create a double stranded break, which in anembodiment is the presence of two nickase activities; an endonucleaseactivity; an exonuclease activity; and a helicase activity, i.e., theability to unwind the helical structure of a double stranded nucleicacid.

In an embodiment, an enzymatically active Cas9 molecule cleaves both DNAstrands and results in a double stranded break. In an embodiment, a Cas9molecule cleaves only one strand, e.g., the strand to which the gRNAhybridizes to, or the strand complementary to the strand the gRNAhybridizes with. In an embodiment, an active Cas9 molecule comprisescleavage activity associated with an HNH-like domain. In an embodiment,an active Cas9 molecule comprises cleavage activity associated with anN-terminal RuvC-like domain. In an embodiment, an active Cas9 moleculecomprises cleavage activity associated with an HNH-like domain andcleavage activity associated with an N-terminal RuvC-like domain. In anembodiment, an active Cas9 molecule comprises an active, or cleavagecompetent, HNH-like domain and an inactive, or cleavage incompetent,N-terminal RuvC-like domain. In an embodiment, an active Cas9 moleculecomprises an inactive, or cleavage incompetent, HNH-like domain and anactive, or cleavage competent, N-terminal RuvC-like domain.

In an embodiment, the ability of an active Cas9 molecule to interactwith and cleave a target nucleic acid is PAM sequence dependent. A PAMsequence is a sequence in the target nucleic acid. In an embodiment,cleavage of the target nucleic acid occurs upstream from the PAMsequence. Active Cas9 molecules from different bacterial species canrecognize different sequence motifs (e.g., PAM sequences). In anembodiment, an active Cas9 molecule of S. pyogenes recognizes thesequence motif NGG and directs cleavage of a target nucleic acidsequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence.See, e.g., Mali el ai, SCIENCE 2013; 339(6121): 823-826. In anembodiment, an active Cas9 molecule of S. thermophilus recognizes thesequence motif NGGNG and NNAG AAW (W=A or T) and directs cleavage of acore target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairsupstream from these sequences. See, e.g., Horvath et al., SCIENCE 2010;327(5962): 167-170, and Deveau et al, J BACTERIOL 2008; 190(4):1390-1400. In an embodiment, an active Cas9 molecule of S mulansrecognizes the sequence motif NGG or NAAR (R-A or G) and directscleavage of a core target nucleic acid sequence 1 to 10, e.g., 3 to 5base pairs, upstream from this sequence. See, e.g., Deveau et al., JBACTERIOL 2008; 190(4): 1 390-1400.

In an embodiment, an active Cas9 molecule of S. aureus recognizes thesequence motif NNGRR (R=A or G) and directs cleavage of a target nucleicacid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from thatsequence. See, e.g., Ran F. et al., NATURE, vol. 520, 2015, pp. 186-191.In an embodiment, an active Cas9 molecule of N. meningitidis recognizesthe sequence motif NNNNGATT and directs cleavage of a target nucleicacid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from thatsequence. See, e.g., Hou et al., PNAS EARLY EDITION 2013, 1-6. Theability of a Cas9 molecule to recognize a PAM sequence can bedetermined, e.g., using a transformation assay described in Jinek et al,SCIENCE 2012, 337:816.

Some Cas9 molecules have the ability to interact with a gRNA molecule,and in conjunction with the gRNA molecule home (e.g., targeted orlocalized) to a core target domain, but are incapable of cleaving thetarget nucleic acid, or incapable of cleaving at efficient rates. Cas9molecules having no, or no substantial, cleavage activity may bereferred to herein as an inactive Cas9 (an enzymatically inactive Cas9),a dead Cas9, or a dCas9 molecule. For example, an inactive Cas9 moleculecan lack cleavage activity or have substantially less, e.g., less than20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9molecule, as measured by an assay described herein.

Exemplary naturally occurring Cas9 molecules are described in Chylinskiet al, RNA Biology 2013; 10:5, 727-737. Such Cas9 molecules include Cas9molecules of a cluster 1 bacterial family, cluster 2 bacterial family,cluster 3 bacterial family, cluster 4 bacterial family, cluster 5bacterial family, cluster 6 bacterial family, a cluster 7 bacterialfamily, a cluster 8 bacterial family, a cluster 9 bacterial family, acluster 10 bacterial family, a cluster 11 bacterial family, a cluster 12bacterial family, a cluster 13 bacterial family, a cluster 14 bacterialfamily, a cluster 1 bacterial family, a cluster 16 bacterial family, acluster 17 bacterial family, a cluster 18 bacterial family, a cluster 19bacterial family, a cluster 20 bacterial family, a cluster 21 bacterialfamily, a cluster 22 bacterial family, a cluster 23 bacterial family, acluster 24 bacterial family, a cluster 25 bacterial family, a cluster 26bacterial family, a cluster 27 bacterial family, a cluster 28 bacterialfamily, a cluster 29 bacterial family, a cluster 30 bacterial family, acluster 31 bacterial family, a cluster 32 bacterial family, a cluster 33bacterial family, a cluster 34 bacterial family, a cluster 35 bacterialfamily, a cluster 36 bacterial family, a cluster 37 bacterial family, acluster 38 bacterial family, a cluster 39 bacterial family, a cluster 40bacterial family, a cluster 41 bacterial family, a cluster 42 bacterialfamily, a cluster 43 bacterial family, a cluster 44 bacterial family, acluster 45 bacterial family, a cluster 46 bacterial family, a cluster 47bacterial family, a cluster 48 bacterial family, a cluster 49 bacterialfamily, a cluster 50 bacterial family, a cluster 51 bacterial family, acluster 52 bacterial family, a cluster 53 bacterial family, a cluster 54bacterial family, a cluster 55 bacterial family, a cluster 56 bacterialfamily, a cluster 57 bacterial family, a cluster 58 bacterial family, acluster 59 bacterial family, a cluster 60 bacterial family, a cluster 61bacterial family, a cluster 62 bacterial family, a cluster 63 bacterialfamily, a cluster 64 bacterial family, a cluster 65 bacterial family, acluster 66 bacterial family, a cluster 67 bacterial family, a cluster 68bacterial family, a cluster 69 bacterial family, a cluster 70 bacterialfamily, a cluster 71 bacterial family, a cluster 72 bacterial family, acluster 73 bacterial family, a cluster 74 bacterial family, a cluster 75bacterial family, a cluster 76 bacterial family, a cluster 77 bacterialfamily, or a cluster 78 bacterial family.

Exemplary naturally occurring Cas9 molecules include a Cas9 molecule ofa cluster 1 bacterial family. Examples include a Cas9 molecule of: S.pyogenes (e.g., strain SF370, MGAS 10270, MGAS 10750, MGAS2096, MGAS315,MGAS5005, MGAS6180, MGAS9429, NZ131 and SSI-1), S. thermophilus (e.g.,strain LMD-9), S. pseudoporcinus (e.g., strain SPIN 20026), S. mutans(e.g., strain UA 159, NN2025), S. macacae (e.g., strain NCTC1 1558), S.gallolylicus (e.g., strain UCN34, ATCC BAA-2069), S. equines (e.g.,strain ATCC 9812, MGCS 124), S. dysdalactiae (e.g., strain GGS 124), S.bovis (e.g., strain ATCC 700338), S. cmginosus (e.g.; strain F021 1), S.agalactia* (e.g., strain NEM316, A909), Listeria monocytogenes (e.g.,strain F6854), Listeria innocua (L. innocua, e.g., strain Clip 1 1262),Etuerococcus italicus (e.g., strain DSM 15952), or Enterococcus faecium(e.g., strain 1,231,408). Additional exemplary Cas9 molecules are a Cas9molecule of Neisseria meningitidis (Hou et al. PNAS Early Edition 2013,1-6) and a S. aureus Cas9 molecule.

In an embodiment, a Cas9 molecule, e.g., an active Cas9 molecule orinactive Cas9 molecule, comprises an amino acid sequence: having 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with;differs at no more than 1%, 2%, 5%, 10%, 15%, 20%, 30%, or 40% of theamino acid residues when compared with; differs by at least 1, 2, 5, 10or 20 amino acids but by no more than 100, 80, 70, 60, 50, 40 or 30amino acids from; or is identical to; any Cas9 molecule sequencedescribed herein or a naturally occurring Cas9 molecule sequence, e.g.,a Cas9 molecule from a species listed herein or described in Chylinskiet al., RNA Biology 2013, 10:5,Γ2Γ-T,1 Hou et al. PNAS Early Edition2013, 1-6.

In an embodiment, a Cas9 molecule comprises an amino acid sequencehaving 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%homology with; differs at no more than 1%, 2%, 5%, 10%, 15%, 20%, 30%,or 40% of the amino acid residues when compared with; differs by atleast 1, 2, 5, 10 or 20 amino acids but by no more than 100, 80, 70, 60,50, 40 or 30 amino acids from; or is identical to; S. pyogenes Cas9:

(SEQ ID NO: 205) Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr AsnSer Val 1               5                   10                  15 GlyTrp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe            20                  25                  30 Lys Val Leu GlyAsn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile        35                  40                  45 Gly Ala Leu Leu PheAsp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu    50                  55                  60 Lys Arg Thr Ala Arg ArgArg Tyr Thr Arg Arg Lys Asn Arg Ile Cys65                  70                  75                  80 Tyr LeuGln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser                85                  90                  95 Phe Phe HisArg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys            100                 105                 110 His Glu Arg HisPro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr        115                 120                 125 His Glu Lys Tyr ProThr Ile Tyr His Leu Arg Lys Lys Leu Val Asp    130                 135                 140 Ser Thr Asp Lys Ala AspLeu Arg Leu Ile Tyr Leu Ala Leu Ala His145                 150                 155                 160 Met IleLys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro                165                 170                 175 Asp Asn SerAsp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr            180                 185                 190 Asn Gln Leu PheGlu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala        195                 200                 205 Lys Ala Ile Leu SerAla Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn    210                 215                 220 Leu Ile Ala Gln Leu ProGly Glu Lys Lys Asn Gly Leu Phe Gly Asn225                 230                 235                 240 Leu IleAla Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe                245                 250                 255 Asp Leu AlaGlu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp            260                 265                 270 Asp Asp Leu AspAsn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp        275                 280                 285 Leu Phe Leu Ala AlaLys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp    290                 295                 300 Ile Leu Arg Val Asn ThrGlu Ile Thr Lys Ala Pro Leu Ser Ala Ser305                 310                 315                 320 Met IleLys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys                325                 330                 335 Ala Leu ValArg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe            340                 345                 350 Asp Gln Ser LysAsn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser        355                 360                 365 Gln Glu Glu Phe TyrLys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp    370                 375                 380 Gly Thr Glu Glu Leu LeuVal Lys Leu Asn Arg Glu Asp Leu Leu Arg385                 390                 395                 400 Lys GlnArg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu                405                 410                 415 Gly Glu LeuHis Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe            420                 425                 430 Leu Lys Asp AsnArg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile        435                 440                 445 Pro Tyr Tyr Val GlyPro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp    450                 455                 460 Met Thr Arg Lys Ser GluGlu Thr Ile Thr Pro Trp Asn Phe Glu Glu465                 470                 475                 480 Val ValAsp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr                485                 490                 495 Asn Phe AspLys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser            500                 505                 510 Leu Leu Tyr GluTyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys        515                 520                 525 Tyr Val Thr Glu GlyMet Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln    530                 535                 540 Lys Lys Ala Ile Val AspLeu Leu Phe Lys Thr Asn Arg Lys Val Thr545                 550                 555                 560 Val LysGln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp                565                 570                 575 Ser Val GluIle Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly            580                 585                 590 Thr Tyr His AspLeu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp        595                 600                 605 Asn Glu Glu Asn GluAsp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr    610                 615                 620 Leu Phe Glu Asp Arg GluMet Ile Glu Glu Arg Leu Lys Thr Tyr Ala625                 630                 635                 640 His LeuPhe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr                645                 650                 655 Thr Gly TrpGly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp            660                 665                 670 Lys Gln Ser GlyLys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe        675                 680                 685 Ala Asn Arg Asn PheMet Gln Leu Ile His Asp Asp Ser Leu Thr Phe    690                 695                 700 Lys Glu Asp Ile Gln LysAla Gln Val Ser Gly Gln Gly Asp Ser Leu705                 710                 715                 720 His GluHis Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly                725                 730                 735 Ile Leu GlnThr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly            740                 745                 750 Arg His Lys ProGlu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln        755                 760                 765 Thr Thr Gln Lys GlyGln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile    770                 775                 780 Glu Glu Gly Ile Lys GluLeu Gly Ser Gln Ile Leu Lys Glu His Pro785                 790                 795                 800 Val GluAsn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu                805                 810                 815 Gln Asn GlyArg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg            820                 825                 830 Leu Ser Asp TyrAsp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys        835                 840                 845 Asp Asp Ser Ile AspAsn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg    850                 855                 860 Gly Lys Ser Asp Asn ValPro Ser Glu Glu Val Val Lys Lys Met Lys865                 870                 875                 880 Asn TyrTrp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys                885                 890                 895 Phe Asp AsnLeu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp            900                 905                 910 Lys Ala Gly PheIle Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr        915                 920                 925 Lys His Val Ala GlnIle Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp    930                 935                 940 Glu Asn Asp Lys Leu IleArg Glu Val Lys Val Ile Thr Leu Lys Ser945                 950                 955                 960 Lys LeuVal Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg                965                 970                 975 Glu Ile AsnAsn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val            980                 985                 990 Val Gly Thr AlaLeu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe        995                 1000                1005 Val Tyr Gly Asp TyrLys Val Tyr Asp Val Arg Lys Met Ile Ala Lys    1010                1015                1020 Ser Glu Gln Glu Ile GlyLys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser1025                1030                1035                1040 Asn IleMet Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu                1045                1050                1055 Ile Arg LysArg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile            1060                1065                1070 Val Trp Asp LysGly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser        1075                1080                1085 Met Pro Gln Val AsnIle Val Lys Lys Thr Glu Val Gln Thr Gly Gly    1090                1095                1100 Phe Ser Lys Glu Ser IleLeu Pro Lys Arg Asn Ser Asp Lys Leu Ile1105                1110                1115                1120 Ala ArgLys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser                1125                1130                1135 Pro Thr ValAla Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly            1140                1145                1150 Lys Ser Lys LysLeu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile        1155                1160                1165 Met Glu Arg Ser SerPhe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala    1170                1175                1180 Lys Gly Tyr Lys Glu ValLys Lys Asp Leu Ile Ile Lys Leu Pro Lys1185                1190                1195                1200 Tyr SerLeu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser                1205                1210                1215 Ala Gly GluLeu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr            1220                1225                1230 Val Asn Phe LeuTyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser        1235                1240                1245 Pro Glu Asp Asn GluGln Lys Gln Leu Phe Val Glu Gln His Lys His    1250                1255                1260 Tyr Leu Asp Glu Ile IleGlu Gln Ile Ser Glu Phe Ser Lys Arg Val1265                1270                1275                1280 Ile LeuAla Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys                1285                1290                1295 His Arg AspLys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu            1300                1305                1310 Phe Thr Leu ThrAsn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp        1315                1320                1325 Thr Thr Ile Asp ArgLys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp    1330                1335                1340 Ala Thr Leu Ile His GlnSer Ile Thr Gly Leu Tyr Glu Thr Arg Ile1345                1350                1355                1360 Asp LeuSer Gln Leu Gly Gly Asp                 1365

In embodiments, the Cas9 molecule is a S. pyogenes Cas9 variant of SEQID NO: 205 that includes one or more mutations to positively chargedamino acids (e.g., lysine, arginine or histidine) that introduce anuncharged or nonpolar amino acid, e.g., alanine, at said position. Inembodiments, the mutation is to one or more positively charged aminoacids in the nt-groove of Cas9. In embodiments, the Cas9 molecule is aS. pyogenes Cas9 variant of SEQ ID NO: 205 that includes a mutation atposition 855 of SEQ ID NO: 205, for example a mutation to an unchargedamino acid, e.g., alanine, at position 855 of SEQ ID NO: 205. Inembodiments, the Cas9 molecule has a mutation only at position 855 ofSEQ ID NO: 205, relative to SEQ ID NO: 205, e.g., to an uncharged aminoacid, e.g., alanine. In embodiments, the Cas9 molecule is a S. pyogenesCas9 variant of SEQ ID NO: 205 that includes a mutation at position 810,a mutation at position 1003, and/or a mutation at position 1060 of SEQID NO: 205, for example a mutation to alanine at position 810, position1003, and/or position 1060 of SEQ ID NO: 205. In embodiments, the Cas9molecule has a mutation only at position 810, position 1003, andposition 1060 of SEQ ID NO: 205, relative to SEQ ID NO: 205, e.g., whereeach mutation is to an uncharged amino acid, for example, alanine. Inembodiments, the Cas9 molecule is a S. pyogenes Cas9 variant of SEQ IDNO: 205 that includes a mutation at position 848, a mutation at position1003, and/or a mutation at position 1060 of SEQ ID NO: 205, for examplea mutation to alanine at position 848, position 1003, and/or position1060 of SEQ ID NO: 205. In embodiments, the Cas9 molecule has a mutationonly at position 848, position 1003, and position 1060 of SEQ ID NO:205, relative to SEQ ID NO: 205, e.g., where each mutation is to anuncharged amino acid, for example, alanine. In embodiments, the Cas9molecule is a Cas9 molecule as described in Slaymaker et al., ScienceExpress, available online Dec. 1, 2015 at Science DOI:10.1126/science.aad5227.

In embodiments, the Cas9 molecule is a S. pyogenes Cas9 variant of SEQID NO: 205 that includes one or more mutations. In embodiments, the Cas9variant comprises a mutation at position 80 of SEQ ID NO: 205, e.g.,includes a leucine at position 80 of SEQ ID NO: 205 (i.e., comprises,e.g., consists of, SEQ ID NO: 205 with a C80L mutation). In embodiments,the Cas9 variant comprises a mutation at position 574 of SEQ ID NO: 205,e.g., includes a glutamic acid at position 574 of SEQ ID NO: 205 (i.e.,comprises, e.g., consists of, SEQ ID NO: 205 with a C574E mutation). Inembodiments, the Cas9 variant comprises a mutation at position 80 and amutation at position 574 of SEQ ID NO: 205, e.g., includes a leucine atposition 80 of SEQ ID NO: 205, and a glutamic acid at position 574 ofSEQ ID NO: 205 (i.e., comprises, e.g., consists of, SEQ ID NO: 205 witha C80L mutation and a C574E mutation). Without being bound by theory, itis believed that such mutations improve the solution properties of theCas9 molecule.

In embodiments, the Cas9 molecule is a S. pyogenes Cas9 variant of SEQID NO: 205 that includes one or more mutations. In embodiments, the Cas9variant comprises a mutation at position 147 of SEQ ID NO: 205, e.g.,includes a tyrosine at position 147 of SEQ ID NO: 205 (i.e., comprises,e.g., consists of, SEQ ID NO: 205 with a D147Y mutation). Inembodiments, the Cas9 variant comprises a mutation at position 411 ofSEQ ID NO: 205, e.g., includes a threonine at position 411 of SEQ ID NO:205 (i.e., comprises, e.g., consists of, SEQ ID NO: 205 with a P411Tmutation). In embodiments, the Cas9 variant comprises a mutation atposition 147 and a mutation at position 411 of SEQ ID NO: 205, e.g.,includes a tyrosine at position 147 of SEQ ID NO: 205, and a threonineat position 411 of SEQ ID NO: 205 (i.e., comprises, e.g., consists of,SEQ ID NO: 205 with a D147Y mutation and a P411T mutation). Withoutbeing bound by theory, it is believed that such mutations improve thetargeting efficiency of the Cas9 molecule, e.g., in yeast.

In embodiments, the Cas9 molecule is a S. pyogenes Cas9 variant of SEQID NO: 205 that includes one or more mutations. In embodiments, the Cas9variant comprises a mutation at position 1135 of SEQ ID NO: 205, e.g.,includes a glutamic acid at position 1135 of SEQ ID NO: 205 (i.e.,comprises, e.g., consists of, SEQ ID NO: 205 with a D1135E mutation).Without being bound by theory, it is believed that such mutationsimprove the selectivity of the Cas9 molecule for the NGG PAM sequenceversus the NAG PAM sequence.

In embodiments, the Cas9 molecule is a S. pyogenes Cas9 variant of SEQID NO: 205 that includes one or more mutations that introduce anuncharged or nonpolar amino acid, e.g., alanine, at certain positions.In embodiments, the Cas9 molecule is a S. pyogenes Cas9 variant of SEQID NO: 205 that includes a mutation at position 497, a mutation atposition 661, a mutation at position 695 and/or a mutation at position926 of SEQ ID NO: 205, for example a mutation to alanine at position497, position 661, position 695 and/or position 926 of SEQ ID NO: 205.In embodiments, the Cas9 molecule has a mutation only at position 497,position 661, position 695, and position 926 of SEQ ID NO: 205, relativeto SEQ ID NO: 205, e.g., where each mutation is to an uncharged aminoacid, for example, alanine Without being bound by theory, it is believedthat such mutations reduce the cutting by the Cas9 molecule atoff-target sites

It will be understood that the mutations described herein to the Cas9molecule may be combined, and may be combined with any of the fusions orother modifications described herein, and the Cas9 molecule tested inthe assays described herein.

Various types of Cas molecules can be used to practice the inventionsdisclosed herein. In some embodiments, Cas molecules of Type II Cassystems are used. In other embodiments, Cas molecules of other Cassystems are used. For example, Type I or Type III Cas molecules may beused. Exemplary Cas molecules (and Cas systems) are described, e.g., inHaft et ai, PLoS COMPUTATIONAL BIOLOGY 2005, 1(6): e60 and Makarova etal, NATURE REVIEW MICROBIOLOGY 2011, 9:467-477, the contents of bothreferences are incorporated herein by reference in their entirety.

In an embodiment, the Cas9 molecule comprises one or more of thefollowing activities: a nickase activity; a double stranded cleavageactivity (e.g., an endonuclease and/or exonuclease activity); a helicaseactivity; or the ability, together with a gRNA molecule, to localize toa target nucleic acid.

Altered Cas9 Molecules

Naturally occurring Cas9 molecules possess a number of properties,including: nickase activity, nuclease activity (e.g., endonucleaseand/or exonuclease activity); helicase activity; the ability toassociate functionally with a gRNA molecule; and the ability to target(or localize to) a site on a nucleic acid (e.g., PAM recognition andspecificity). In an embodiment, a Cas9 molecules can include all or asubset of these properties. In typical embodiments, Cas9 molecules havethe ability to interact with a gRNA molecule and, in concert with thegRNA molecule, localize to a site in a nucleic acid. Other activities,e.g., PAM specificity, cleavage activity, or helicase activity can varymore widely in Cas9 molecules.

Cas9 molecules with desired properties can be made in a number of ways,e.g., by alteration of a parental, e.g., naturally occurring Cas9molecules to provide an altered Cas9 molecule having a desired property.For example, one or more mutations or differences relative to a parentalCas9 molecule can be introduced. Such mutations and differencescomprise: substitutions (e.g., conservative substitutions orsubstitutions of non-essential amino acids); insertions; or deletions.In an embodiment, a Cas9 molecule can comprises one or more mutations ordifferences, e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50mutations but less than 200, 100, or 80 mutations relative to areference Cas9 molecule.

In an embodiment, a mutation or mutations do not have a substantialeffect on a Cas9 activity, e.g. a Cas9 activity described herein. In anembodiment, a mutation or mutations have a substantial effect on a Cas9activity, e.g. a Cas9 activity described herein. In an embodiment,exemplary activities comprise one or more of PAM specificity, cleavageactivity, and helicase activity. A mutation(s) can be present, e.g., in:one or more RuvC-like domain, e.g., an N-terminal RuvC-like domain; anHNH-like domain; a region outside the RuvC-like domains and the HNH-likedomain. In some embodiments, a mutation(s) is present in an N-terminalRuvC-like domain. In some embodiments, a mutation(s) is present in anHNH-like domain. In some embodiments, mutations are present in both anN-terminal RuvC-like domain and an HNH-like domain.

Whether or not a particular sequence, e.g., a substitution, may affectone or more activity, such as targeting activity, cleavage activity,etc, can be evaluated or predicted, e.g., by evaluating whether themutation is conservative or by the method described in Section III. Inan embodiment, a “non-essential” amino acid residue, as used in thecontext of a Cas9 molecule, is a residue that can be altered from thewild-type sequence of a Cas9 molecule, e.g., a naturally occurring Cas9molecule, e.g., an active Cas9 molecule, without abolishing or morepreferably, without substantially altering a Cas9 activity (e.g.,cleavage activity), whereas changing an “essential” amino acid residueresults in a substantial loss of activity (e.g., cleavage activity).

Cas9 Molecules with Altered PAM Recognition or No PAM Recognition

Naturally occurring Cas9 molecules can recognize specific PAM sequences,for example the PAM recognition sequences described above for S.pyogenes, S. thermophilus, S. mutans, S. aureus and N. meningitidis.

In an embodiment, a Cas9 molecule has the same PAM specificities as anaturally occurring Cas9 molecule. In other embodiments, a Cas9 moleculehas a PAM specificity not associated with a naturally occurring Cas9molecule, or a PAM specificity not associated with the naturallyoccurring Cas9 molecule to which it has the closest sequence homology.For example, a naturally occurring Cas9 molecule can be altered, e.g.,to alter PAM recognition, e.g., to alter the PAM sequence that the Cas9molecule recognizes to decrease off target sites and/or improvespecificity; or eliminate a PAM recognition requirement. In anembodiment, a Cas9 molecule can be altered, e.g., to increase length ofPAM recognition sequence and/or improve Cas9 specificity to high levelof identity to decrease off target sites and increase specificity. In anembodiment, the length of the PAM recognition sequence is at least 4, 5,6, 7, 8, 9, 10 or 15 amino acids in length. Cas9 molecules thatrecognize different PAM sequences and/or have reduced off-targetactivity can be generated using directed evolution. Exemplary methodsand systems that can be used for directed evolution of Cas9 moleculesare described, e.g., in Esvelt el al, Nature 2011, 472(7344): 499-503.Candidate Cas9 molecules can be evaluated, e.g., by methods describedherein.

Non-Cleaving and Modified-Cleavage Cas9 Molecules

In an embodiment, a Cas9 molecule comprises a cleavage property thatdiffers from naturally occurring Cas9 molecules, e.g., that differs fromthe naturally occurring Cas9 molecule having the closest homology. Forexample, a Cas9 molecule can differ from naturally occurring Cas9molecules, e.g., a Cas9 molecule of S. pyogenes, as follows: its abilityto modulate, e.g., decreased or increased, cleavage of a double strandedbreak (endonuclease and/or exonuclease activity), e.g., as compared to anaturally occurring Cas9 molecule (e.g., a Cas9 molecule of S.pyogenes); its ability to modulate, e.g., decreased or increased,cleavage of a single strand of a nucleic acid, e.g., a non-complimentarystrand of a nucleic acid molecule or a complementary strand of a nucleicacid molecule (nickase activity), e.g., as compared to a naturallyoccurring Cas9 molecule (e.g., a Cas9 molecule of S. pyogenes); or theability to cleave a nucleic acid molecule, e.g., a double stranded orsingle stranded nucleic acid molecule, can be eliminated.

Modified Cleavage active Cas9 Molecules

In an embodiment, an active Cas9 molecule comprises one or more of thefollowing activities: cleavage activity associated with an N-terminalRuvC-like domain; cleavage activity associated with an HNH-like domain;cleavage activity associated with an HNH domain and cleavage activityassociated with an N-terminal RuvC-like domain.

In an embodiment, the Cas9 molecule is a Cas9 nickase, e.g., cleavesonly a single strand of DNA. In an embodiment, the Cas9 nickase includesa mutation at position 10 and/or a mutation at position 840 of SEQ IDNO: 205, e.g., comprises a D10A and/or H840A mutation to SEQ ID NO: 205.

Non-Cleaving Inactive Cas9 Molecules

In an embodiment, the altered Cas9 molecule is an inactive Cas9 moleculewhich does not cleave a nucleic acid molecule (either double stranded orsingle stranded nucleic acid molecules) or cleaves a nucleic acidmolecule with significantly less efficiency, e.g., less than 20, 10, 5,1 or 0.1% of the cleavage activity of a reference Cas9 molecule, e.g.,as measured by an assay described herein. The reference Cas9 moleculecan by a naturally occurring unmodified Cas9 molecule, e.g., a naturallyoccurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, S.thermophilus, S. aureus or N. meningitidis. In an embodiment, thereference Cas9 molecule is the naturally occurring Cas9 molecule havingthe closest sequence identity or homology. In an embodiment, theinactive Cas9 molecule lacks substantial cleavage activity associatedwith an N-terminal RuvC-like domain and cleavage activity associatedwith an HNH-like domain.

In an embodiment, the Cas9 molecule is dCas9. Tsai et al. (2014), Nat.Biotech. 32:569-577.

A catalytically inactive Cas9 molecule may be fused with a transcriptionrepressor. An inactive Cas9 fusion protein complexes with a gRNA andlocalizes to a DNA sequence specified by gRNA's targeting domain, but,unlike an active Cas9, it will not cleave the target DNA. Fusion of aneffector domain, such as a transcriptional repression domain, to aninactive Cas9 enables recruitment of the effector to any DNA sitespecified by the gRNA. Site specific targeting of a Cas9 fusion proteinto a promoter region of a gene can block or affect polymerase binding tothe promoter region, for example, a Cas9 fusion with a transcriptionfactor (e.g., a transcription activator) and/or a transcriptionalenhancer binding to the nucleic acid to increase or inhibittranscription activation. Alternatively, site specific targeting of aCas9-fusion to a transcription repressor to a promoter region of a genecan be used to decrease transcription activation.

Transcription repressors or transcription repressor domains that may befused to an inactive Cas9 molecule can include ruppel associated box(KRAB or SKD), the Mad mSIN3 interaction domain (SID) or the ERFrepressor domain (ERD).

In another embodiment, an inactive Cas9 molecule may be fused with aprotein that modifies chromatin. For example, an inactive Cas9 moleculemay be fused to heterochromatin protein 1 (HP1), a histone lysinemethyltransferase (e.g., SUV39H 1, SUV39H2, G9A, ESET/SETDB 1,Pr-SET7/8, SUV4-20H 1,RIZ1), a histone lysine demethylates (e.g.,LSD1/BHC1 10, SpLsdl/Sw, 1/Safl 10, Su(var)3-3, JMJD2A/JHDM3A, JMJD2B,JMJD2C/GASC1, JMJD2D, Rphl, JARID 1 A/RBP2, JARI DIB/PLU-I, JAR1D1C/SMCX, JARID1 D/SMCY, Lid, Jhn2, Jmj2), a histone lysine deacetylases(e.g., HDAC1, HDAC2, HDAC3, HDAC8, Rpd3, Hos 1, Cir6, HDAC4, HDAC5,HDAC7, HDAC9, Hdal, Cir3, SIRT 1, SIRT2, Sir2, Hst 1, Hst2, Hst3, Hst4,HDAC 11) and a DNA methylases (DNMTLDNMT2a/DMNT3b, MET1). An inactiveCas9-chomatin modifying molecule fusion protein can be used to alterchromatin status to reduce expression a target gene.

The heterologous sequence (e.g., the transcription repressor domain) maybe fused to the N- or C-terminus of the inactive Cas9 protein. In analternative embodiment, the heterologous sequence (e.g., thetranscription repressor domain) may be fused to an internal portion(i.e., a portion other than the N-terminus or C-terminus) of theinactive Cas9 protein.

The ability of a Cas9 molecule/gRNA molecule complex to bind to andcleave a target nucleic acid can be evaluated, e.g., by the methodsdescribed herein in Section III. The activity of a Cas9 molecule, e.g.,either an active Cas9 or a inactive Cas9, alone or in a complex with agRNA molecule may also be evaluated by methods well-known in the art,including, gene expression assays and chromatin-based assays, e.g.,chromatin immunoprecipitation (ChiP) and chromatin in vivo assay (CiA).

Other Cas9 Molecule Fusions

In embodiments, the Cas9 molecule, e.g, a Cas9 of S. pyogenes, mayadditionally comprise one or more amino acid sequences that conferadditional activity.

In some aspects, the Cas9 molecule may comprise one or more nuclearlocalization sequences (NLSs), such as at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more NLSs. In some embodiments, the Cas9 molecule comprises atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near theamino-terminus, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs ator near the carboxy-terminus, or a combination of these (e.g. one ormore NLS at the amino-terminus and one or more NLS at the carboxyterminus). When more than one NLS is present, each may be selectedindependently of the others, such that a single NLS may be present inmore than one copy and/or in combination with one or more other NLSspresent in one or more copies. In some embodiments, an NLS is considerednear the N- or C-terminus when the nearest amino acid of the NLS iswithin about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more aminoacids along the polypeptide chain from the N- or C-terminus. Typically,an NLS consists of one or more short sequences of positively chargedlysines or arginines exposed on the protein surface, but other types ofNLS are known. Non-limiting examples of NLSs include an NLS sequencecomprising or derived from: the NLS of the SV40 virus large T-antigen,having the amino acid sequence PKKKRKV (SEQ ID NO: 206); the NLS fromnucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequenceKRPAATKKAGQAKKKK (SEQ ID NO: 207); the c-myc NLS having the amino acidsequence PAAKRVKLD (SEQ ID NO: 208) or RQRRNELKRSP (SEQ ID NO: 209); thehRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY(SEQ ID NO: 210); the sequenceRMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 211) of the IBBdomain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 212) andPPKKARED (SEQ ID NO: 213) of the myoma T protein; the sequence PQPKKKPL(SEQ ID NO: 214) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO:215) of mouse c-ab1 IV; the sequences DRLRR (SEQ ID NO: 216) and PKQKKRK(SEQ ID NO: 217) of the influenza virus NS1; the sequence RKLKKKIKKL(SEQ ID NO: 218) of the Hepatitis virus delta antigen; the sequenceREKKKFLKRR (SEQ ID NO: 219) of the mouse Mx1 protein; the sequenceKRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 220) of the human poly(ADP-ribose)polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 221) of thesteroid hormone receptors (human) glucocorticoid. Other suitable NLSsequences are known in the art (e.g., Sorokin, Biochemistry (Moscow)(2007) 72:13, 1439-1457; Lange J Biol Chem. (2007) 282:8, 5101-5).

In an embodiment, the Cas9 molecule, e.g., S. pyogenes Cas9 molecule,comprises a NLS sequence of SV40, e.g., disposed N terminal to the Cas9molecule. In an embodiment, the Cas9 molecule, e.g., S. pyogenes Cas9molecule, comprises a NLS sequence of SV40 disposed N-terminal to theCas9 molecule and a NLS sequence of SV40 disposed C terminal to the Cas9molecule. In an embodiment, the Cas9 molecule, e.g., S. pyogenes Cas9molecule, comprises a NLS sequence of SV40 disposed N-terminal to theCas9 molecule and a NLS sequence of nucleoplasmin disposed C-terminal tothe Cas9 molecule. In any of the aforementioned embodiments, themolecule may additionally comprise a tag, e.g., a His tag, e.g., aHis(6) tag (SEQ ID NO: 247) or His(8) tag (SEQ ID NO: 248), e.g., at theN terminus or the C terminus.

In some aspects, the Cas9 molecule may comprise one or more amino acidsequences that allow the Cas9 molecule to be specifically recognized,for example a tag. In one embodiment, the tag is a Histidine tag, e.g.,a histidine tag comprising at least 3, 4, 5, 6, 7, 8, 9, 10 or morehistidine amino acids. In embodiments, the histidine tag is a His6 tag(six histidines) (SEQ ID NO: 247). In other embodiments, the histidinetag is a His8 tag (eight histidines) (SEQ ID NO: 248). In embodiments,the histidine tag may be separated from one or more other portions ofthe Cas9 molecule by a linker. In embodiments, the linker is GGS. Anexample of such a fusion is the Cas9 molecule iProt106520.

In some aspects, the Cas9 molecule may comprise one or more amino acidsequences that are recognized by a protease (e.g., comprise a proteasecleavage site). In embodiments, the cleavage site is the tobacco etchvirus (TEV) cleavage site, e.g., comprises the sequence ENLYFQG (SEQ IDNO: 230). In some aspects the protease cleavage site, e.g., the TEVcleavage site is disposed between a tag, e.g., a His tag, e.g., a His6(SEQ ID NO: 247) or His8 tag (SEQ ID NO: 248), and the remainder of theCas9 molecule. Without being bound by theory it is believed that suchintroduction will allow for the use of the tag for, e.g., purificationof the Cas9 molecule, and then subsequent cleavage so the tag does notinterfere with the Cas9 molecule function.

In embodiments, the Cas9 molecule (e.g., a Cas9 molecule as describedherein) comprises an N-terminal NLS, and a C-terminal NLS (e.g.,comprises, from N- to C-terminal NLS-Cas9-NLS), e.g., wherein each NLSis an SV40 NLS (PKKKRKV (SEQ ID NO: 206)). In embodiments, the Cas9molecule (e.g., a Cas9 molecule as described herein) comprises anN-terminal NLS, a C-terminal NLS, and a C-terminal His6 tag (SEQ ID NO:247) (e.g., comprises, from N- to C-terminal NLS-Cas9-NLS-His tag),e.g., wherein each NLS is an SV40 NLS (PKKKRKV (SEQ ID NO: 206)). Inembodiments, the Cas9 molecule (e.g., a Cas9 molecule as describedherein) comprises an N-terminal His tag (e.g., His6 tag (SEQ ID NO:247)), an N-terminal NLS, and a C-terminal NLS (e.g., comprises, from N-to C-terminal His tag-NLS-Cas9-NLS), e.g., wherein each NLS is an SV40NLS (PKKKRKV (SEQ ID NO: 206)). In embodiments, the Cas9 molecule (e.g.,a Cas9 molecule as described herein) comprises an N-terminal NLS and aC-terminal His tag (e.g., His6 tag (SEQ ID NO: 247)) (e.g., comprisesfrom N- to C-terminal His tag-Cas9-NLS), e.g., wherein the NLS is anSV40 NLS (PKKKRKV (SEQ ID NO: 206)). In embodiments, the Cas9 molecule(e.g., a Cas9 molecule as described herein) comprises an N-terminal NLSand a C-terminal His tag (e.g., His6 tag (SEQ ID NO: 247)) (e.g.,comprises from N- to C-terminal NLS-Cas9-His tag), e.g., wherein the NLSis an SV40 NLS (PKKKRKV (SEQ ID NO: 206)). In embodiments, the Cas9molecule (e.g., a Cas9 molecule as described herein) comprises anN-terminal His tag (e.g., His8 tag (SEQ ID NO: 248)), an N-terminalcleavage domain (e.g., a tobacco etch virus (TEV) cleavage domain (e.g.,comprises the sequence ENLYFQG (SEQ ID NO: 230))), an N-terminal NLS(e.g., an SV40 NLS; SEQ ID NO: 206), and a C-terminal NLS (e.g., an SV40NLS; SEQ ID NO: 206) (e.g., comprises from N- to C-terminal Histag-TEV-NLS-Cas9-NLS). In any of the aforementioned embodiments the Cas9has the sequence of SEQ ID NO: 205. Alternatively, in any of theaforementioned embodiments, the Cas9 has a sequence of a Cas9 variant ofSEQ ID NO: 205, e.g., as described herein. In any of the aforementionedembodiments, the Cas9 molecule comprises a linker between the His tagand another portion of the molecule, e.g., a GGS linker. Amino acidsequences of exemplary Cas9 molecules described above are providedbelow. “iProt” identifiers match those in FIG. 60.

iProt105026 (also referred to as iProt106154, iProt106331, iProt106545,and PID426303, depending on the preparation of the protein) (SEQ ID NO:233): MAPKKKRKVD KKYSIGLDIG TNSVGWAVIT DEYKVPSKKF KVLGNTDRHS IKKNLIGALLFDSGETAEAT RLKRTARRRY TRRKNRICYL QEIFSNEMAK VDDSFFHRLE ESFLVEEDKKHERHPIFGNI VDEVAYHEKY PTIYHLRKKL VDSTDKADLR LIYLALAHMI KFRGHFLIEGDLNPDNSDVD KLFIQLVQTY NQLFEENPIN ASGVDAKAIL SARLSKSRRL ENLIAQLPGEKKNGLFGNLI ALSLGLTPNF KSNFDLAEDA KLQLSKDTYD DDLDNLLAQI GDQYADLFLAAKNLSDAILL SDILRVNTEI TKAPLSASMI KRYDEHHQDL TLLKALVRQQ LPEKYKEIFFDQSKNGYAGY IDGGASQEEF YKFIKPILEK MDGTEELLVK LNREDLLRKQ RTFDNGSIPHQIHLGELHAI LRRQEDFYPF LKDNREKIEK ILTFRIPYYV GPLARGNSRF AWMTRKSEETITPWNFEEVV DKGASAQSFI ERMTNFDKNL PNEKVLPKHS LLYEYFTVYN ELTKVKYVTEGMRKPAFLSG EQKKAIVDLL FKTNRKVTVK QLKEDYFKKI ECFDSVEISG VEDRFNASLGTYHDLLKIIK DKDFLDNEEN EDILEDIVLT LTLFEDREMI EERLKTYAHL FDDKVMKQLKRRRYTGWGRL SRKLINGIRD KQSGKTILDF LKSDGFANRN FMQLIHDDSL TFKEDIQKAQVSGQGDSLHE HIANLAGSPA IKKGILQTVK VVDELVKVMG RHKPENIVIE MARENQTTQKGQKNSRERMK RIEEGIKELG SQILKEHPVE NTQLQNEKLY LYYLQNGRDM YVDQELDINRLSDYDVDHIV PQSFLKDDSI DNKVLTRSDK NRGKSDNVPS EEVVKKMKNY WRQLLNAKLITQRKFDNLTK AERGGLSELD KAGFIKRQLV ETRQITKHVA QILDSRMNTK YDENDKLIREVKVITLKSKL VSDFRKDFQF YKVREINNYH HAHDAYLNAV VGTALIKKYP KLESEFVYGDYKVYDVRKMI AKSEQEIGKA TAKYFFYSNI MNFFKTEITL ANGEIRKRPL IETNGETGEIVWDKGRDFAT VRKVLSMPQV NIVKKTEVQT GGFSKESILP KRNSDKLIAR KKDWDPKKYGGFDSPTVAYS VLVVAKVEKG KSKKLKSVKE LLGITIMERS SFEKNPIDFL EAKGYKEVKKDLIIKLPKYS LFELENGRKR MLASAGELQK GNELALPSKY VNFLYLASHY EKLKGSPEDNEQKQLFVEQH KHYLDEIIEQ ISEFSKRVIL ADANLDKVLS AYNKHRDKPI REQAENIIHLFTLTNLGAPA AFKYFDTTID RKRYTSTKEV LDATLIHQSI TGLYETRIDL SQLGGDSRADPKKKRKVHHH HHH iProt106518 (SEQ ID NO: 234): MAPKKKRKVD KKYSIGLDIGTNSVGWAVIT DEYKVPSKKF KVLGNTDRHS IKKNLIGALL FDSGETAEAT RLKRTARRRYTRRKNRILYL QEIFSNEMAK VDDSFFHRLE ESFLVEEDKK HERHPIFGNI VDEVAYHEKYPTIYHLRKKL VDSTDKADLR LIYLALAHMI KFRGHFLIEG DLNPDNSDVD KLFIQLVQTYNQLFEENPIN ASGVDAKAIL SARLSKSRRL ENLIAQLPGE KKNGLFGNLI ALSLGLTPNFKSNFDLAEDA KLQLSKDTYD DDLDNLLAQI GDQYADLFLA AKNLSDAILL SDILRVNTEITKAPLSASMI KRYDEHHQDL TLLKALVRQQ LPEKYKEIFF DQSKNGYAGY IDGGASQEEFYKFIKPILEK MDGTEELLVK LNREDLLRKQ RTFDNGSIPH QIHLGELHAI LRRQEDFYPFLKDNREKIEK ILTFRIPYYV GPLARGNSRF AWMTRKSEET ITPWNFEEVV DKGASAQSFIERMTNFDKNL PNEKVLPKHS LLYEYFTVYN ELTKVKYVTE GMRKPAFLSG EQKKAIVDLLFKTNRKVTVK QLKEDYFKKI EEFDSVEISG VEDRFNASLG TYHDLLKIIK DKDFLDNEENEDILEDIVLT LTLFEDREMI EERLKTYAHL FDDKVMKQLK RRRYTGWGRL SRKLINGIRDKQSGKTILDF LKSDGFANRN FMQLIHDDSL TFKEDIQKAQ VSGQGDSLHE HIANLAGSPAIKKGILQTVK VVDELVKVMG RHKPENIVIE MARENQTTQK GQKNSRERMK RIEEGIKELGSQILKEHPVE NTQLQNEKLY LYYLQNGRDM YVDQELDINR LSDYDVDHIV PQSFLKDDSIDNKVLTRSDK NRGKSDNVPS EEVVKKMKNY WRQLLNAKLI TQRKFDNLTK AERGGLSELDKAGFIKRQLV ETRQITKHVA QILDSRMNTK YDENDKLIRE VKVITLKSKL VSDFRKDFQFYKVREINNYH HAHDAYLNAV VGTALIKKYP KLESEFVYGD YKVYDVRKMI AKSEQEIGKATAKYFFYSNI MNFFKTEITL ANGEIRKRPL IETNGETGEI VWDKGRDFAT VRKVLSMPQVNIVKKTEVQT GGFSKESILP KRNSDKLIAR KKDWDPKKYG GFDSPTVAYS VLVVAKVEKGKSKKLKSVKE LLGITIMERS SFEKNPIDFL EAKGYKEVKK DLIIKLPKYS LFELENGRKRMLASAGELQK GNELALPSKY VNFLYLASHY EKLKGSPEDN EQKQLFVEQH KHYLDEIIEQISEFSKRVIL ADANLDKVLS AYNKHRDKPI REQAENIIHL FTLTNLGAPA AFKYFDTTIDRKRYTSTKEV LDATLIHQSI TGLYETRIDL SQLGGDSRAD PKKKRKVHHH HHH iProt106519(SEQ ID NO: 235): MGSSHHHHHH HHENLYFQGS MDKKYSIGLD IGTNSVGWAV ITDEYKVPSKKFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNEMAKVDDSFFHR LEESFLVEED KKHERHPIFG NIVDEVAYHE KYPTIYHLRK KLVDSTDKADLRLIYLALAH MIKFRGHFLI EGDLNPDNSD VDKLFIQLVQ TYNQLFEENP INASGVDAKAILSARLSKSR RLENLIAQLP GEKKNGLFGN LIALSLGLTP NFKSNFDLAE DAKLQLSKDTYDDDLDNLLA QIGDQYADLF LAAKNLSDAI LLSDILRVNT EITKAPLSAS MIKRYDEHHQDLTLLKALVR QQLPEKYKEI FFDQSKNGYA GYIDGGASQE EFYKFIKPIL EKMDGTEELLVKLNREDLLR KQRTFDNGSI PHQIHLGELH AILRRQEDFY PFLKDNREKI EKILTFRIPYYVGPLARGNS RFAWMTRKSE ETITPWNFEE VVDKGASAQS FIERMTNFDK NLPNEKVLPKHSLLYEYFTV YNELTKVKYV TEGMRKPAFL SGEQKKAIVD LLFKTNRKVT VKQLKEDYFKKIECFDSVEI SGVEDRFNAS LGTYHDLLKI IKDKDFLDNE ENEDILEDIV LTLTLFEDREMIEERLKTYA HLFDDKVMKQ LKRRRYTGWG RLSRKLINGI RDKQSGKTIL DFLKSDGFANRNFMQLIHDD SLTFKEDIQK AQVSGQGDSL HEHIANLAGS PAIKKGILQT VKVVDELVKVMGRHKPENIV IEMARENQTT QKGQKNSRER MKRIEEGIKE LGSQILKEHP VENTQLQNEKLYLYYLQNGR DMYVDQELDI NRLSDYDVDH IVPQSFLKDD SIDNKVLTRS DKNRGKSDNVPSEEVVKKMK NYWRQLLNAK LITQRKFDNL TKAERGGLSE LDKAGFIKRQ LVETRQITKHVAQILDSRMN TKYDENDKLI REVKVITLKS KLVSDFRKDF QFYKVREINN YHHAHDAYLNAVVGTALIKK YPKLESEFVY GDYKVYDVRK MIAKSEQEIG KATAKYFFYS NIMNFFKTEITLANGEIRKR PLIETNGETG EIWVDKGRDF ATVRKVLSMP QVNIVKKTEV QTGGFSKESILPKRNSDKLI ARKKDWDPKK YGGFDSPTVA YSVLVVAKVE KGKSKKLKSV KELLGITIMERSSFEKNPID FLEAKGYKEV KKDLIIKLPK YSLFELENGR KRMLASAGEL QKGNELALPSKYVNFLYLAS HYEKLKGSPE DNEQKQLFVE QHKHYLDEII EQISEFSKRV ILADANLDKVLSAYNKHRDK PIREQAENII HLFTLTNLGA PAAFKYFDTT IDRKRYTSTK EVLDATLIHQSITGLYETRI DLSQLGGDGG GSPKKKRKV iProt106520 (SEQ ID NO: 236): MAHHHHHHGGSPKKKRKVDK KYSIGLDIGT NSVGWAVITD EYKVPSKKFK VLGNTDRHSI KKNLIGALLFDSGETAEATR LKRTARRRYT RRKNRICYLQ EIFSNEMAKV DDSFFHRLEE SFLVEEDKKHERHPIFGNIV DEVAYHEKYP TIYHLRKKLV DSTDKADLRL IYLALAHMIK FRGHFLIEGDLNPDNSDVDK LFIQLVQTYN QLFEENPINA SGVDAKAILS ARLSKSRRLE NLIAQLPGEKKNGLFGNLIA LSLGLTPNFK SNFDLAEDAK LQLSKDTYDD DLDNLLAQIG DQYADLFLAAKNLSDAILLS DILRVNTEIT KAPLSASMIK RYDEHHQDLT LLKALVRQQL PEKYKEIFFDQSKNGYAGYI DGGASQEEFY KFIKPILEKM DGTEELLVKL NREDLLRKQR TFDNGSIPHQIHLGELHAIL RRQEDFYPFL KDNREKIEKI LTFRIPYYVG PLARGNSRFA WMTRKSEETITPWNFEEVVD KGASAQSFIE RMTNFDKNLP NEKVLPKHSL LYEYFTVYNE LTKVKYVTEGMRKPAFLSGE QKKAIVDLLF KTNRKVTVKQ LKEDYFKKIE CFDSVEISGV EDRFNASLGTYHDLLKIIKD KDFLDNEENE DILEDIVLTL TLFEDREMIE ERLKTYAHLF DDKVMKQLKRRRYTGWGRLS RKLINGIRDK QSGKTILDFL KSDGFANRNF MQLIHDDSLT FKEDIQKAQVSGQGDSLHEH IANLAGSPAI KKGILQTVKV VDELVKVMGR HKPENIVIEM ARENQTTQKGQKNSRERMKR IEEGIKELGS QILKEHPVEN TQLQNEKLYL YYLQNGRDMY VDQELDINRLSDYDVDHIVP QSFLKDDSID NKVLTRSDKN RGKSDNVPSE EVVKKMKNYW RQLLNAKLITQRKFDNLTKA ERGGLSELDK AGFIKRQLVE TRQITKHVAQ ILDSRMNTKY DENDKLIREVKVITLKSKLV SDFRKDFQFY KVREINNYHH AHDAYLNAVV GTALIKKYPK LESEFVYGDYKVYDVRKMIA KSEQEIGKAT AKYFFYSNIM NFFKTEITLA NGEIRKRPLI ETNGETGEIVWDKGRDFATV RKVLSMPQVN IVKKTEVQTG GFSKESILPK RNSDKLIARK KDWDPKKYGGFDSPTVAYSV LVVAKVEKGK SKKLKSVKEL LGITIMERSS FEKNPIDFLE AKGYKEVKKDLIIKLPKYSL FELENGRKRM LASAGELQKG NELALPSKYV NFLYLASHYE KLKGSPEDNEQKQLFVEQHK HYLDEIIEQI SEFSKRVILA DANLDKVLSA YNKHRDKPIR EQAENIIHLFTLTNLGAPAA FKYFDTTIDR KRYTSTKEVL DATLIHQSIT GLYETRIDLS QLGGDSRADP KKKRKViProt106521 (SEQ ID NO: 237): MAPKKKRKVD KKYSIGLDIG TNSVGWAVITDEYKVPSKKF KVLGNTDRHS IKKNLIGALL FDSGETAEAT RLKRTARRRY TRRKNRICYLQEIFSNEMAK VDDSFFHRLE ESFLVEEDKK HERHPIFGNI VDEVAYHEKY PTIYHLRKKLVDSTDKADLR LIYLALAHMI KFRGHFLIEG DLNPDNSDVD KLFIQLVQTY NQLFEENPINASGVDAKAIL SARLSKSRRL ENLIAQLPGE KKNGLFGNLI ALSLGLTPNF KSNFDLAEDAKLQLSKDTYD DDLDNLLAQI GDQYADLFLA AKNLSDAILL SDILRVNTEI TKAPLSASMIKRYDEHHQDL TLLKALVRQQ LPEKYKEIFF DQSKNGYAGY IDGGASQEEF YKFIKPILEKMDGTEELLVK LNREDLLRKQ RTFDNGSIPH QIHLGELHAI LRRQEDFYPF LKDNREKIEKILTFRIPYYV GPLARGNSRF AWMTRKSEET ITPWNFEEVV DKGASAQSFI ERMTNFDKNLPNEKVLPKHS LLYEYFTVYN ELTKVKYVTE GMRKPAFLSG EQKKAIVDLL FKTNRKVTVKQLKEDYFKKI ECFDSVEISG VEDRFNASLG TYHDLLKIIK DKDFLDNEEN EDILEDIVLTLTLFEDREMI EERLKTYAHL FDDKVMKQLK RRRYTGWGRL SRKLINGIRD KQSGKTILDFLKSDGFANRN FMQLIHDDSL TFKEDIQKAQ VSGQGDSLHE HIANLAGSPA IKKGILQTVKVVDELVKVMG RHKPENIVIE MARENQTTQK GQKNSRERMK RIEEGIKELG SQILKEHPVENTQLQNEKLY LYYLQNGRDM YVDQELDINR LSDYDVDHIV PQSFLKDDSI DNKVLTRSDKNRGKSDNVPS EEVVKKMKNY WRQLLNAKLI TQRKFDNLTK AERGGLSELD KAGFIKRQLVETRQITKHVA QILDSRMNTK YDENDKLIRE VKVITLKSKL VSDFRKDFQF YKVREINNYHHAHDAYLNAV VGTALIKKYP KLESEFVYGD YKVYDVRKMI AKSEQEIGKA TAKYFFYSNIMNFFKTEITL ANGEIRKRPL IETNGETGEI VWDKGRDFAT VRKVLSMPQV NIVKKTEVQTGGFSKESILP KRNSDKLIAR KKDWDPKKYG GFDSPTVAYS VLVVAKVEKG KSKKLKSVKELLGITIMERS SFEKNPIDFL EAKGYKEVKK DLIIKLPKYS LFELENGRKR MLASAGELQKGNELALPSKY VNFLYLASHY EKLKGSPEDN EQKQLFVEQH KHYLDEIIEQ ISEFSKRVILADANLDKVLS AYNKHRDKPI REQAENIIHL FTLTNLGAPA AFKYFDTTID RKRYTSTKEVLDATLIHQSI TGLYETRIDL SQLGGDSRAD HHHHHH iProt106522 (SEQ ID NO: 238):MAHHHHHHGG SDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGALLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR LEESFLVEEDKKHERHPIFG NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH MIKFRGHFLIEGDLNPDNSD VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLPGEKKNGLFGN LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLFLAAKNLSDAI LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR QQLPEKYKEIFFDQSKNGYA GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR KQRTFDNGSIPHQIHLGELH AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKSEETITPWNFEE VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYVTEGMRKPAFL SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNASLGTYHDLLKI IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQLKRRRYTGWG RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD SLTFKEDIQKAQVSGQGDSL HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV IEMARENQTTQKGQKNSRER MKRIEEGIKE LGSQILKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDINRLSDYDVDH IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNAKLITQRKFDNL TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLIREVKVITLKS KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK YPKLESEFVYGDYKVYDVRK MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR PLIETNGETGEIVWDKGRDF ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLI ARKKDWDPKKYGGFDSPTVA YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID FLEAKGYKEVKKDLIIKLPK YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS HYEKLKGSPEDNEQKQLFVE QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK PIREQAENIIHLFTLTNLGA PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGDSRADPKKKRKV iProt106658 (SEQ ID NO: 239): MGSSHHHHHH HHENLYFQGS MDKKYSIGLDIGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRTARRRYTRRKNRIC YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG NIVDEVAYHEKYPTIYHLRK KLVDSTDKAD LRLIYLALAH MIKFRGHFLI EGDLNPDNSD VDKLFIQLVQTYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN LIALSLGLTPNFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLF LAAKNLSDAI LLSDILRVNTEITKAPLSAS MIKRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA GYIDGGASQEEFYKFIKPIL EKMDGTEELL VKLNREDLLR KQRTFDNGSI PHQIHLGELH AILRRQEDFYPFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE VVDKGASAQSFIERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL SGEQKKAIVDLLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI IKDKDFLDNEENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQ LKRRRYTGWG RLSRKLINGIRDKQSGKTIL DFLKSDGFAN RNFMQLIHDD SLTFKEDIQK AQVSGQGDSL HEHIANLAGSPAIKKGILQT VKVVDELVKV MGRHKPENIV IEMARENQTT QKGQKNSRER MKRIEEGIKELGSQILKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDH IVPQSFLKDDSIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNAK LITQRKFDNL TKAERGGLSELDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS KLVSDFRKDFQFYKVREINN YHHAHDAYLN AVVGTALIKK YPKLESEFVY GDYKVYDVRK MIAKSEQEIGKATAKYFFYS NIMNFFKTEI TLANGEIRKR PLIETNGETG EIWVDKGRDF ATVRKVLSMPQVNIVKKTEV QTGGFSKESI LPKRNSDKLI ARKKDWDPKK YGGFDSPTVA YSVLVVAKVEKGKSKKLKSV KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK YSLFELENGRKRMLASAGEL QKGNELALPS KYVNFLYLAS HYEKLKGSPE DNEQKQLFVE QHKHYLDEIIEQISEFSKRV ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA PAAFKYFDTTIDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGDGG GSPKKKRKV iProt106745 (SEQID NO: 240): MAPKKKRKVD KKYSIGLDIG TNSVGWAVIT DEYKVPSKKF KVLGNTDRHSIKKNLIGALL FDSGETAEAT RLKRTARRRY TRRKNRICYL QEIFSNEMAK VDDSFFHRLEESFLVEEDKK HERHPIFGNI VDEVAYHEKY PTIYHLRKKL VDSTDKADLR LIYLALAHMIKFRGHFLIEG DLNPDNSDVD KLFIQLVQTY NQLFEENPIN ASGVDAKAIL SARLSKSRRLENLIAQLPGE KKNGLFGNLI ALSLGLTPNF KSNFDLAEDA KLQLSKDTYD DDLDNLLAQIGDQYADLFLA AKNLSDAILL SDILRVNTEI TKAPLSASMI KRYDEHHQDL TLLKALVRQQLPEKYKEIFF DQSKNGYAGY IDGGASQEEF YKFIKPILEK MDGTEELLVK LNREDLLRKQRTFDNGSIPH QIHLGELHAI LRRQEDFYPF LKDNREKIEK ILTFRIPYYV GPLARGNSRFAWMTRKSEET ITPWNFEEVV DKGASAQSFI ERMTNFDKNL PNEKVLPKHS LLYEYFTVYNELTKVKYVTE GMRKPAFLSG EQKKAIVDLL FKTNRKVTVK QLKEDYFKKI ECFDSVEISGVEDRFNASLG TYHDLLKIIK DKDFLDNEEN EDILEDIVLT LTLFEDREMI EERLKTYAHLFDDKVMKQLK RRRYTGWGRL SRKLINGIRD KQSGKTILDF LKSDGFANRN FMQLIHDDSLTFKEDIQKAQ VSGQGDSLHE HIANLAGSPA IKKGILQTVK VVDELVKVMG RHKPENIVIEMARENQTTQK GQKNSRERMK RIEEGIKELG SQILKEHPVE NTQLQNEKLY LYYLQNGRDMYVDQELDINR LSDYDVDHIV PQSFLKDDSI DNAVLTRSDK NRGKSDNVPS EEVVKKMKNYWRQLLNAKLI TQRKFDNLTK AERGGLSELD KAGFIKRQLV ETRQITKHVA QILDSRMNTKYDENDKLIRE VKVITLKSKL VSDFRKDFQF YKVREINNYH HAHDAYLNAV VGTALIKKYPKLESEFVYGD YKVYDVRKMI AKSEQEIGKA TAKYFFYSNI MNFFKTEITL ANGEIRKRPLIETNGETGEI VWDKGRDFAT VRKVLSMPQV NIVKKTEVQT GGFSKESILP KRNSDKLIARKKDWDPKKYG GFDSPTVAYS VLVVAKVEKG KSKKLKSVKE LLGITIMERS SFEKNPIDFLEAKGYKEVKK DLIIKLPKYS LFELENGRKR MLASAGELQK GNELALPSKY VNFLYLASHYEKLKGSPEDN EQKQLFVEQH KHYLDEIIEQ ISEFSKRVIL ADANLDKVLS AYNKHRDKPIREQAENIIHL FTLTNLGAPA AFKYFDTTID RKRYTSTKEV LDATLIHQSI TGLYETRIDLSQLGGDSRAD PKKKRKVHHH HHH iProt106746 (SEQ ID NO: 241): MAPKKKRKVDKKYSIGLDIG TNSVGWAVIT DEYKVPSKKF KVLGNTDRHS IKKNLIGALL FDSGETAEATRLKRTARRRY TRRKNRICYL QEIFSNEMAK VDDSFFHRLE ESFLVEEDKK HERHPIFGNIVDEVAYHEKY PTIYHLRKKL VDSTDKADLR LIYLALAHMI KFRGHFLIEG DLNPDNSDVDKLFIQLVQTY NQLFEENPIN ASGVDAKAIL SARLSKSRRL ENLIAQLPGE KKNGLFGNLIALSLGLTPNF KSNFDLAEDA KLQLSKDTYD DDLDNLLAQI GDQYADLFLA AKNLSDAILLSDILRVNTEI TKAPLSASMI KRYDEHHQDL TLLKALVRQQ LPEKYKEIFF DQSKNGYAGYIDGGASQEEF YKFIKPILEK MDGTEELLVK LNREDLLRKQ RTFDNGSIPH QIHLGELHAILRRQEDFYPF LKDNREKIEK ILTFRIPYYV GPLARGNSRF AWMTRKSEET ITPWNFEEVVDKGASAQSFI ERMTNFDKNL PNEKVLPKHS LLYEYFTVYN ELTKVKYVTE GMRKPAFLSGEQKKAIVDLL FKTNRKVTVK QLKEDYFKKI ECFDSVEISG VEDRFNASLG TYHDLLKIIKDKDFLDNEEN EDILEDIVLT LTLFEDREMI EERLKTYAHL FDDKVMKQLK RRRYTGWGRLSRKLINGIRD KQSGKTILDF LKSDGFANRN FMQLIHDDSL TFKEDIQKAQ VSGQGDSLHEHIANLAGSPA IKKGILQTVK VVDELVKVMG RHKPENIVIE MARENQTTQK GQKNSRERMKRIEEGIKELG SQILKEHPVE NTQLQNEALY LYYLQNGRDM YVDQELDINR LSDYDVDHIVPQSFLKDDSI DNKVLTRSDK NRGKSDNVPS EEVVKKMKNY WRQLLNAKLI TQRKFDNLTKAERGGLSELD KAGFIKRQLV ETRQITKHVA QILDSRMNTK YDENDKLIRE VKVITLKSKLVSDFRKDFQF YKVREINNYH HAHDAYLNAV VGTALIKKYP ALESEFVYGD YKVYDVRKMIAKSEQEIGKA TAKYFFYSNI MNFFKTEITL ANGEIRKAPL IETNGETGEI VWDKGRDFATVRKVLSMPQV NIVKKTEVQT GGFSKESILP KRNSDKLIAR KKDWDPKKYG GFDSPTVAYSVLVVAKVEKG KSKKLKSVKE LLGITIMERS SFEKNPIDFL EAKGYKEVKK DLIIKLPKYSLFELENGRKR MLASAGELQK GNELALPSKY VNFLYLASHY EKLKGSPEDN EQKQLFVEQHKHYLDEIIEQ ISEFSKRVIL ADANLDKVLS AYNKHRDKPI REQAENIIHL FTLTNLGAPAAFKYFDTTID RKRYTSTKEV LDATLIHQSI TGLYETRIDL SQLGGDSRAD PKKKRKVHHH HHHiProt106747 (SEQ ID NO: 242): MAPKKKRKVD KKYSIGLDIG TNSVGWAVITDEYKVPSKKF KVLGNTDRHS IKKNLIGALL FDSGETAEAT RLKRTARRRY TRRKNRICYLQEIFSNEMAK VDDSFFHRLE ESFLVEEDKK HERHPIFGNI VDEVAYHEKY PTIYHLRKKLVDSTDKADLR LIYLALAHMI KFRGHFLIEG DLNPDNSDVD KLFIQLVQTY NQLFEENPINASGVDAKAIL SARLSKSRRL ENLIAQLPGE KKNGLFGNLI ALSLGLTPNF KSNFDLAEDAKLQLSKDTYD DDLDNLLAQI GDQYADLFLA AKNLSDAILL SDILRVNTEI TKAPLSASMIKRYDEHHQDL TLLKALVRQQ LPEKYKEIFF DQSKNGYAGY IDGGASQEEF YKFIKPILEKMDGTEELLVK LNREDLLRKQ RTFDNGSIPH QIHLGELHAI LRRQEDFYPF LKDNREKIEKILTFRIPYYV GPLARGNSRF AWMTRKSEET ITPWNFEEVV DKGASAQSFI ERMTNFDKNLPNEKVLPKHS LLYEYFTVYN ELTKVKYVTE GMRKPAFLSG EQKKAIVDLL FKTNRKVTVKQLKEDYFKKI ECFDSVEISG VEDRFNASLG TYHDLLKIIK DKDFLDNEEN EDILEDIVLTLTLFEDREMI EERLKTYAHL FDDKVMKQLK RRRYTGWGRL SRKLINGIRD KQSGKTILDFLKSDGFANRN FMQLIHDDSL TFKEDIQKAQ VSGQGDSLHE HIANLAGSPA IKKGILQTVKVVDELVKVMG RHKPENIVIE MARENQTTQK GQKNSRERMK RIEEGIKELG SQILKEHPVENTQLQNEKLY LYYLQNGRDM YVDQELDINR LSDYDVDHIV PQSFLADDSI DNKVLTRSDKNRGKSDNVPS EEVVKKMKNY WRQLLNAKLI TQRKFDNLTK AERGGLSELD KAGFIKRQLVETRQITKHVA QILDSRMNTK YDENDKLIRE VKVITLKSKL VSDFRKDFQF YKVREINNYHHAHDAYLNAV VGTALIKKYP ALESEFVYGD YKVYDVRKMI AKSEQEIGKA TAKYFFYSNIMNFFKTEITL ANGEIRKAPL IETNGETGEI VWDKGRDFAT VRKVLSMPQV NIVKKTEVQTGGFSKESILP KRNSDKLIAR KKDWDPKKYG GFDSPTVAYS VLVVAKVEKG KSKKLKSVKELLGITIMERS SFEKNPIDFL EAKGYKEVKK DLIIKLPKYS LFELENGRKR MLASAGELQKGNELALPSKY VNFLYLASHY EKLKGSPEDN EQKQLFVEQH KHYLDEIIEQ ISEFSKRVILADANLDKVLS AYNKHRDKPI REQAENIIHL FTLTNLGAPA AFKYFDTTID RKRYTSTKEVLDATLIHQSI TGLYETRIDL SQLGGDSRAD PKKKRKVHHH HHH iProt106884 (SEQ ID NO:243): MAPKKKRKVD KKYSIGLDIG TNSVGWAVIT DEYKVPSKKF KVLGNTDRHS IKKNLIGALLFDSGETAEAT RLKRTARRRY TRRKNRICYL QEIFSNEMAK VDDSFFHRLE ESFLVEEDKKHERHPIFGNI VDEVAYHEKY PTIYHLRKKL VDSTDKADLR LIYLALAHMI KFRGHFLIEGDLNPDNSDVD KLFIQLVQTY NQLFEENPIN ASGVDAKAIL SARLSKSRRL ENLIAQLPGEKKNGLFGNLI ALSLGLTPNF KSNFDLAEDA KLQLSKDTYD DDLDNLLAQI GDQYADLFLAAKNLSDAILL SDILRVNTEI TKAPLSASMI KRYDEHHQDL TLLKALVRQQ LPEKYKEIFFDQSKNGYAGY IDGGASQEEF YKFIKPILEK MDGTEELLVK LNREDLLRKQ RTFDNGSIPHQIHLGELHAI LRRQEDFYPF LKDNREKIEK ILTFRIPYYV GPLARGNSRF AWMTRKSEETITPWNFEEVV DKGASAQSFI ERMTAFDKNL PNEKVLPKHS LLYEYFTVYN ELTKVKYVTEGMRKPAFLSG EQKKAIVDLL FKTNRKVTVK QLKEDYFKKI ECFDSVEISG VEDRFNASLGTYHDLLKIIK DKDFLDNEEN EDILEDIVLT LTLFEDREMI EERLKTYAHL FDDKVMKQLKRRRYTGWGAL SRKLINGIRD KQSGKTILDF LKSDGFANRN FMALIHDDSL TFKEDIQKAQVSGQGDSLHE HIANLAGSPA IKKGILQTVK VVDELVKVMG RHKPENIVIE MARENQTTQKGQKNSRERMK RIEEGIKELG SQILKEHPVE NTQLQNEKLY LYYLQNGRDM YVDQELDINRLSDYDVDHIV PQSFLKDDSI DNKVLTRSDK NRGKSDNVPS EEVVKKMKNY WRQLLNAKLITQRKFDNLTK AERGGLSELD KAGFIKRQLV ETRAITKHVA QILDSRMNTK YDENDKLIREVKVITLKSKL VSDFRKDFQF YKVREINNYH HAHDAYLNAV VGTALIKKYP KLESEFVYGDYKVYDVRKMI AKSEQEIGKA TAKYFFYSNI MNFFKTEITL ANGEIRKRPL IETNGETGEIVWDKGRDFAT VRKVLSMPQV NIVKKTEVQT GGFSKESILP KRNSDKLIAR KKDWDPKKYGGFDSPTVAYS VLVVAKVEKG KSKKLKSVKE LLGITIMERS SFEKNPIDFL EAKGYKEVKKDLIIKLPKYS LFELENGRKR MLASAGELQK GNELALPSKY VNFLYLASHY EKLKGSPEDNEQKQLFVEQH KHYLDEIIEQ ISEFSKRVIL ADANLDKVLS AYNKHRDKPI REQAENIIHLFTLTNLGAPA AFKYFDTTID RKRYTSTKEV LDATLIHQSI TGLYETRIDL SQLGGDSRADPKKKRKVHHH HHH iProt 20109496 (SEQ ID NO: 244)MAPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRILYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIEEFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSRADHHHHHH

Nucleic Acids Encoding Cas9 Molecules

Nucleic acids encoding the Cas9 molecules, e.g., an active Cas9 moleculeor an inactive Cas9 molecule are provided herein.

Exemplary nucleic acids encoding Cas9 molecules are described in Cong etal, SCIENCE 2013, 399(6121):819-823; Wang et al, CELL 2013,153(4):910-918; Mali et al., SCIENCE 2013, 399(6121):823-826; Jinek etal, SCIENCE 2012, 337(6096):816-821.

In an embodiment, a nucleic acid encoding a Cas9 molecule can be asynthetic nucleic acid sequence. For example, the synthetic nucleic acidmolecule can be chemically modified, e.g., as described in Section XIII.In an embodiment, the Cas9 mRNA has one or more of, e.g., all of thefollowing properties: it is capped, polyadenylated, substituted with5-methylcytidine and/or pseudouridine.

In addition or alternatively, the synthetic nucleic acid sequence can becodon optimized, e.g., at least one non-common codon or less-commoncodon has been replaced by a common codon. For example, the syntheticnucleic acid can direct the synthesis of an optimized messenger mRNA,e.g., optimized for expression in a mammalian expression system, e.g.,described herein.

Provided below is an exemplary codon optimized nucleic acid sequenceencoding a Cas9 molecule of S. pyogenes.

(SEQ ID NO: 222) atggataaaa agtacagcat cgggctggac atcggtacaa actcagtggggtgggccgtg 60 attacggacg agtacaaggt accctccaaa aaatttaaag tgctgggtaacacggacaga 120 cactctataa agaaaaatct tattggagcc ttgctgttcg actcaggcgagacagccgaa 180 gccacaaggt tgaagcggac cgccaggagg cggtatacca ggagaaagaaccgcatatgc 240 tacctgcaag aaatcttcag taacgagatg gcaaaggttg acgatagctttttccatcgc 300 ctggaagaat cctttcttgt tgaggaagac aagaagcacg aacggcaccccatctttggc 360 aatattgtcg acgaagtggc atatcacgaa aagtacccga ctatctaccacctcaggaag 420 aagctggtgg actctaccga taaggcggac ctcagactta tttatttggcactcgcccac 480 atgattaaat ttagaggaca tttcttgatc gagggcgacc tgaacccggacaacagtgac 540 gtcgataagc tgttcatcca acttgtgcag acctacaatc aactgttcgaagaaaaccct 600 ataaatgctt caggagtcga cgctaaagca atcctgtccg cgcgcctctcaaaatctaga 660 agacttgaga atctgattgc tcagttgccc ggggaaaaga aaaatggattgtttggcaac 720 ctgatcgccc tcagtctcgg actgacccca aatttcaaaa gtaacttcgacctggccgaa 780 gacgctaagc tccagctgtc caaggacaca tacgatgacg acctcgacaatctgctggcc 840 cagattgggg atcagtacgc cgatctcttt ttggcagcaa agaacctgtccgacgccatc 900 ctgttgagcg atatcttgag agtgaacacc gaaattacta aagcaccccttagcgcatct 960 atgatcaagc ggtacgacga gcatcatcag gatctgaccc tgctgaaggctcttgtgagg 1020 caacagctcc ccgaaaaata caaggaaatc ttctttgacc agagcaaaaacggctacgct 1080 ggctatatag atggtggggc cagtcaggag gaattctata aattcatcaagcccattctc 1140 gagaaaatgg acggcacaga ggagttgctg gtcaaactta acagggaggacctgctgcgg 1200 aagcagcgga cctttgacaa cgggtctatc ccccaccaga ttcatctgggcgaactgcac 1260 gcaatcctga ggaggcagga ggatttttat ccttttctta aagataaccgcgagaaaata 1320 gaaaagattc ttacattcag gatcccgtac tacgtgggac ctctcgcccggggcaattca 1380 cggtttgcct ggatgacaag gaagtcagag gagactatta caccttggaacttcgaagaa 1440 gtggtggaca agggtgcatc tgcccagtct ttcatcgagc ggatgacaaattttgacaag 1500 aacctcccta atgagaaggt gctgcccaaa cattctctgc tctacgagtactttaccgtc 1560 tacaatgaac tgactaaagt caagtacgtc accgagggaa tgaggaagccggcattcctt 1620 agtggagaac agaagaaggc gattgtagac ctgttgttca agaccaacaggaaggtgact 1680 gtgaagcaac ttaaagaaga ctactttaag aagatcgaat gttttgacagtgtggaaatt 1740 tcaggggttg aagaccgctt caatgcgtca ttggggactt accatgatcttctcaagatc 1800 ataaaggaca aagacttcct ggacaacgaa gaaaatgagg atattctcgaagacatcgtc 1860 ctcaccctga ccctgttcga agacagggaa atgatagaag agcgcttgaaaacctatgcc 1920 cacctcttcg acgataaagt tatgaagcag ctgaagcgca ggagatacacaggatgggga 1980 agattgtcaa ggaagctgat caatggaatt agggataaac agagtggcaagaccatactg 2040 gatttcctca aatctgatgg cttcgccaat aggaacttca tgcaactgattcacgatgac 2100 tctcttacct tcaaggagga cattcaaaag gctcaggtga gcgggcagggagactccctt 2160 catgaacaca tcgcgaattt ggcaggttcc cccgctatta aaaagggcatccttcaaact 2220 gtcaaggtgg tggatgaatt ggtcaaggta atgggcagac ataagccagaaaatattgtg 2280 atcgagatgg cccgcgaaaa ccagaccaca cagaagggcc agaaaaatagtagagagcgg 2340 atgaagagga tcgaggaggg catcaaagag ctgggatctc agattctcaaagaacacccc 2400 gtagaaaaca cacagctgca gaacgaaaaa ttgtacttgt actatctgcagaacggcaga 2460 gacatgtacg tcgaccaaga acttgatatt aatagactgt ccgactatgacgtagaccat 2520 atcgtgcccc agtccttcct gaaggacgac tccattgata acaaagtcttgacaagaagc 2580 gacaagaaca ggggtaaaag tgataatgtg cctagcgagg aggtggtgaaaaaaatgaag 2640 aactactggc gacagctgct taatgcaaag ctcattacac aacggaagttcgataatctg 2700 acgaaagcag agagaggtgg cttgtctgag ttggacaagg cagggtttattaagcggcag 2760 ctggtggaaa ctaggcagat cacaaagcac gtggcgcaga ttttggacagccggatgaac 2820 acaaaatacg acgaaaatga taaactgata cgagaggtca aagttatcacgctgaaaagc 2880 aagctggtgt ccgattttcg gaaagacttc cagttctaca aagttcgcgagattaataac 2940 taccatcatg ctcacgatgc gtacctgaac gctgttgtcg ggaccgccttgataaagaag 3000 tacccaaagc tggaatccga gttcgtatac ggggattaca aagtgtacgatgtgaggaaa 3060 atgatagcca agtccgagca ggagattgga aaggccacag ctaagtacttcttttattct 3120 aacatcatga atttttttaa gacggaaatt accctggcca acggagagatcagaaagcgg 3180 ccccttatag agacaaatgg tgaaacaggt gaaatcgtct gggataagggcagggatttc 3240 gctactgtga ggaaggtgct gagtatgcca caggtaaata tcgtgaaaaaaaccgaagta 3300 cagaccggag gattttccaa ggaaagcatt ttgcctaaaa gaaactcagacaagctcatc 3360 gcccgcaaga aagattggga ccctaagaaa tacgggggat ttgactcacccaccgtagcc 3420 tattctgtgc tggtggtagc taaggtggaa aaaggaaagt ctaagaagctgaagtccgtg 3480 aaggaactct tgggaatcac tatcatggaa agatcatcct ttgaaaagaaccctatcgat 3540 ttcctggagg ctaagggtta caaggaggtc aagaaagacc tcatcattaaactgccaaaa 3600 tactctctct tcgagctgga aaatggcagg aagagaatgt tggccagcgccggagagctg 3660 caaaagggaa acgagcttgc tctgccctcc aaatatgtta attttctctatctcgcttcc 3720 cactatgaaa agctgaaagg gtctcccgaa gataacgagc agaagcagctgttcgtcgaa 3780 cagcacaagc actatctgga tgaaataatc gaacaaataa gcgagttcagcaaaagggtt 3840 atcctggcgg atgctaattt ggacaaagta ctgtctgctt ataacaagcaccgggataag 3900 cctattaggg aacaagccga gaatataatt cacctcttta cactcacgaatctcggagcc 3960 cccgccgcct tcaaatactt tgatacgact atcgaccgga aacggtataccagtaccaaa 4020 gaggtcctcg atgccaccct catccaccag tcaattactg gcctgtacgaaacacggatc 4080 gacctctctc aactgggcgg cgactag 4107

Provided below is an exemplary codon optimized nucleic acid sequenceencoding a Cas9 molecule including SEQ ID NO: 244:

(SEQ ID NO: 223) ATGGCTCCGAAGAAAAAGCGTAAAGTGGATAAAAAATACAGCATTGGTCTGGACATTGGCACGAACTCAGTGGGTTGGGCGGTCATCACGGATGAATATAAGGTCCCGTCAAAAAAGTTCAAAGTGCTGGGCAACACCGATCGCCATTCGATTAAAAAGAATCTGATCGGCGCGCTGCTGTTTGATAGCGGTGAAACCGCGGAAGCAACGCGTCTGAAACGTACCGCACGTCGCCGTTACACGCGCCGTAAAAATCGTATTCTGTATCTGCAGGAAATCTTTAGCAACGAAATGGCGAAAGTTGATGACTCATTTTTCCACCGCCTGGAAGAATCGTTTCTGGTCGAAGAAGACAAAAAGCATGAACGTCACCCGATTTTCGGTAATATCGTTGATGAAGTCGCGTACCATGAAAAATATCCGACGATTTACCATCTGCGTAAAAAACTGGTGGATTCAACCGACAAAGCCGATCTGCGCCTGATTTACCTGGCACTGGCTCATATGATCAAATTTCGTGGCCACTTCCTGATTGAAGGTGACCTGAACCCGGATAACTCTGACGTTGATAAGCTGTTCATCCAGCTGGTCCAAACCTATAATCAGCTGTTCGAAGAAAACCCGATCAATGCAAGTGGCGTTGATGCGAAGGCCATTCTGTCCGCTCGCCTGAGTAAATCCCGCCGTCTGGAAAACCTGATTGCACAACTGCCGGGCGAAAAGAAAAACGGCCTGTTTGGTAATCTGATCGCTCTGTCACTGGGTCTGACGCCGAACTTTAAATCGAATTTCGACCTGGCAGAAGATGCTAAGCTGCAGCTGAGCAAAGATACCTACGATGACGATCTGGACAACCTGCTGGCGCAAATTGGTGACCAGTATGCCGACCTGTTTCTGGCGGCCAAAAATCTGTCAGATGCCATTCTGCTGTCGGACATCCTGCGCGTGAACACCGAAATCACGAAAGCGCCGCTGTCAGCCTCGATGATTAAACGCTACGATGAACATCACCAGGACCTGACCCTGCTGAAAGCACTGGTTCGTCAGCAACTGCCGGAAAAGTACAAGGAAATTTTCTTTGACCAATCTAAGAACGGCTATGCAGGTTACATCGATGGCGGTGCTAGTCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAAATGGATGGCACGGAAGAACTGCTGGTGAAACTGAATCGTGAAGATCTGCTGCGTAAACAACGCACCTTTGACAACGGCAGCATTCCGCATCAGATCCACCTGGGTGAACTGCATGCGATTCTGCGCCGTCAGGAAGATTTTTATCCGTTCCTGAAAGACAACCGTGAAAAAATTGAAAAGATCCTGACGTTTCGCATCCCGTATTACGTTGGCCCGCTGGCGCGTGGTAATAGCCGCTTCGCCTGGATGACCCGCAAATCTGAAGAAACCATTACGCCGTGGAACTTTGAAGAAGTGGTTGATAAAGGTGCAAGCGCTCAGTCTTTTATCGAACGTATGACCAATTTCGATAAAAACCTGCCGAATGAAAAGGTCCTGCCGAAACATAGCCTGCTGTATGAATACTTTACCGTGTACAACGAACTGACGAAAGTGAAGTATGTTACCGAAGGCATGCGCAAACCGGCGTTTCTGTCTGGTGAACAGAAAAAAGCCATTGTGGATCTGCTGTTCAAGACCAATCGTAAAGTTACGGTCAAACAGCTGAAGGAAGATTACTTCAAAAAGATCGAAGAATTCGACAGCGTGGAAATTTCTGGCGTTGAAGATCGTTTCAACGCCAGTCTGGGTACCTATCATGACCTGCTGAAGATCATCAAGGACAAGGATTTTCTGGATAACGAAGAAAATGAAGACATTCTGGAAGATATCGTGCTGACCCTGACGCTGTTCGAAGATCGTGAAATGATTGAAGAACGCCTGAAAACGTACGCACACCTGTTTGACGATAAAGTTATGAAGCAGCTGAAACGCCGTCGCTATACCGGCTGGGGTCGTCTGTCTCGCAAACTGATTAATGGCATCCGCGATAAGCAAAGTGGTAAAACGATTCTGGATTTCCTGAAATCCGACGGCTTTGCCAACCGTAATTTCATGCAGCTGATCCATGACGATAGTCTGACCTTTAAGGAAGACATTCAGAAAGCACAAGTGTCAGGCCAGGGTGATTCGCTGCATGAACACATTGCGAACCTGGCCGGCTCCCCGGCTATTAAAAAGGGTATCCTGCAGACCGTCAAAGTCGTGGATGAACTGGTGAAGGTTATGGGCCGTCACAAACCGGAAAACATTGTGATCGAAATGGCGCGCGAAAATCAGACCACGCAAAAGGGTCAGAAAAACTCACGTGAACGCATGAAGCGCATTGAAGAAGGCATCAAAGAACTGGGTTCGCAGATTCTGAAAGAACATCCGGTTGAAAACACCCAGCTGCAAAATGAAAAACTGTACCTGTATTACCTGCAAAATGGCCGTGACATGTATGTCGATCAGGAACTGGACATCAACCGCCTGAGCGACTATGATGTCGACCACATTGTGCCGCAGAGCTTTCTGAAGGACGATTCTATCGATAATAAAGTGCTGACCCGTTCTGATAAGAACCGCGGTAAAAGCGACAATGTTCCGTCTGAAGAAGTTGTCAAAAAGATGAAGAACTACTGGCGTCAACTGCTGAATGCGAAGCTGATTACGCAGCGTAAATTCGATAACCTGACCAAGGCGGAACGCGGCGGTCTGAGTGAACTGGATAAGGCCGGCTTTATCAAACGTCAACTGGTGGAAACCCGCCAGATTACGAAACATGTTGCCCAGATCCTGGATTCCCGCATGAACACGAAATATGACGAAAATGATAAGCTGATTCGTGAAGTCAAAGTGATCACCCTGAAGAGTAAGCTGGTGTCCGATTTCCGTAAGGACTTTCAGTTCTACAAAGTTCGCGAAATTAACAATTACCATCACGCACACGATGCTTATCTGAATGCAGTGGTTGGCACCGCTCTGATCAAAAAGTATCCGAAACTGGAAAGCGAATTTGTGTATGGTGATTACAAAGTCTATGACGTGCGCAAGATGATTGCGAAAAGTGAACAGGAAATCGGCAAGGCGACCGCCAAGTACTTTTTCTATTCCAACATCATGAACTTTTTCAAGACCGAAATCACGCTGGCAAATGGCGAAATTCGTAAACGCCCGCTGATCGAAACCAACGGCGAAACGGGTGAAATTGTGTGGGATAAAGGTCGTGACTTCGCGACCGTTCGCAAAGTCCTGTCAATGCCGCAAGTGAATATCGTTAAAAAGACCGAAGTTCAGACGGGCGGTTTTAGTAAAGAATCCATCCTGCCGAAGCGTAACTCGGATAAACTGATTGCGCGCAAAAAGGATTGGGACCCGAAAAAGTACGGCGGTTTTGATAGTCCGACCGTTGCATATTCCGTCCTGGTCGTGGCTAAAGTTGAAAAAGGCAAGAGTAAAAAGCTGAAGTCCGTCAAAGAACTGCTGGGTATTACCATCATGGAACGTAGCTCTTTTGAAAAGAACCCGATTGACTTCCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGATCTGATTATCAAGCTGCCGAAATATTCGCTGTTCGAACTGGAAAACGGTCGTAAACGCATGCTGGCAAGCGCTGGCGAACTGCAGAAGGGTAATGAACTGGCACTGCCGTCTAAATATGTGAACTTTCTGTACCTGGCTAGCCATTATGAAAAACTGAAGGGTTCTCCGGAAGATAACGAACAGAAGCAACTGTTCGTTGAACAACATAAACACTACCTGGATGAAATCATCGAACAGATCTCAGAATTCTCGAAACGCGTCATTCTGGCGGATGCCAATCTGGACAAAGTGCTGAGCGCGTATAACAAGCATCGTGATAAACCGATTCGCGAACAGGCCGAAAATATTATCCACCTGTTTACCCTGACGAACCTGGGCGCACCGGCAGCTTTTAAATACTTCGATACCACGATCGACCGTAAGCGCTATACCAGCACGAAAGAAGTTCTGGATGCTACCCTGATTCATCAGTCAATCACCGGTCTGTATGAAACGCGTATTGACCTGAGCCAACTGGGCGGTGATAGCCGTGCCGACCATCACCATC ACCATCACTAATAG

If the above Cas9 sequences are fused with a peptide or polypeptide atthe C-terminus (e.g., an inactive Cas9 fused with a transcriptionrepressor at the C-terminus), it is understood that the stop codon willbe removed.

Also provided herein are nucleic acids, vectors and cells for productionof a Cas9 molecule, for example a Cas9 molecule described herein. Therecombinant production of polypeptide molecules can be accomplishedusing techniques known to a skilled artisan. Described herein aremolecules and methods for the recombinant production of polypeptidemolecules, such as Cas9 molecules, e.g., as described herein. As used inconnection herewith, “recombinant” molecules and production includes allpolypeptides (e.g., Cas9 molecules, for example as described herein)that are prepared, expressed, created or isolated by recombinant means,such as polypeptides isolated from an animal (e.g., a mouse) that istransgenic or transchromosomal for nucleic acid encoding the molecule ofinterest, a hybridoma prepared therefrom, molecules isolated from a hostcell transformed to express the molecule, e.g., from a transfectoma,molecules isolated from a recombinant, combinatorial library, andmolecules prepared, expressed, created or isolated by any other meansthat involve splicing of all or a portion of a gene encoding themolecule (or portion thereof) to other DNA sequences. Recombinantproduction may be from a host cell, for example, a host cell comprisingnucleic acid encoding a molecule described herein, e.g., a Cas9molecule, e.g., a Cas9 molecule described herein.

Provided herein are nucleic acid molecules encoding a molecule (e.g.,Cas9 molecule and/or gRNA molecule), e.g., as described herein.Specifically provided are nucleic acid molecules comprising sequenceencoding any one of SEQ ID NO: 233 to SEQ ID NO: 244, or encoding afragment of any of SEQ ID NO: 233 to SEQ ID NO: 244, or encoding apolypeptide comprising at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence homology to any of SEQ ID NO: 233 toSEQ ID NO: 244.

Provided herein are vectors, e.g., as described herein, comprising anyof the above-described nucleic acid molecules. In embodiments, saidnucleic acid molecules are operably linked to a promoter, for example apromoter operable in the host cell into which the vector is introduced.

Provided herein are host cells comprising one or more nucleic acidmolecules and/or vectors described herein. In embodiments, the host cellis a prokaryotic host cell. In embodiments, the host cell is aeukaryotic host cell. In embodiments, the host cell is a yeast or E.coli cell. In embodiments, the host cell is a mammalian cell, e.g., ahuman cell. Such host cells may be used for the production of arecombinant molecule described herein, e.g., a Cas9 or gRNA molecule,e.g., as described herein.

VI. Functional Analysis of Candidate Molecules

Candidate Cas9 molecules, candidate gRNA molecules, candidate Cas9molecule/gRNA molecule complexes, can be evaluated by art-known methodsor as described herein. For example, exemplary methods for evaluatingthe endonuclease activity of Cas9 molecule are described, e.g., in Jinekel al., SCIENCE 2012; 337(6096):816-821.

VII. Template Nucleic Acids (for Introduction of Nucleic Acids)

The term “template nucleic acid” or “donor template” as used hereinrefers to a nucleic acid to be inserted at or near a target sequencethat has been modified, e.g., cleaved, by a CRISPR system of the presentinvention. In an embodiment, nucleic acid sequence at or near the targetsequence is modified to have some or all of the sequence of the templatenucleic acid, typically at or near cleavage site(s). In an embodiment,the template nucleic acid is single stranded. In an alternateembodiment, the template nucleic acid is double stranded. In anembodiment, the template nucleic acid is DNA, e.g., double stranded DNA.In an alternate embodiment, the template nucleic acid is single strandedDNA.

In embodiments, the template nucleic acid comprises sequence encoding aglobin protein, e.g., a beta globin, e.g., comprises a beta globin gene.In an embodiment, the beta globin encoded by the nucleic acid comprisesone or more mutations, e.g., anti-sickling mutations. In an embodiment,the beta globin encoded by the nucleic acid comprises the mutation T87Q.In an embodiment, the beta globin encoded by the nucleic acid comprisesthe mutation G16D. In an embodiment, the beta globin encoded by thenucleic acid comprises the mutation E22A. In an embodiment, the betaglobin gene comprises the mutations G16D, E22A and T87Q. In embodiments,the template nucleic acid further comprises one or more regulatoryelements, e.g., a promoter (e.g., a human beta globin promoter), a 3′enhancer, and/or at least a portion of a globin locus control region(e.g., one or more DNAseI hypersensitivity sites (e.g., HS2, HS3 and/orHS4 of the human globin locus)).

In other embodiments, the template nucleic acid comprises sequenceencoding a gamma globin, e.g., comprises a gamma globin gene. Inembodiments, the template nucleic acid comprises sequence encoding morethan one copy of a gamma globin protein, e.g., comprises two or more,e.g., two, gamma globin gene sequences. In embodiments, the templatenucleic acid further comprises one or more regulatory elements, e.g., apromotor and/or enhancer.

In an embodiment, the template nucleic acid alters the structure of thetarget position by participating in a homology directed repair event. Inan embodiment, the template nucleic acid alters the sequence of thetarget position. In an embodiment, the template nucleic acid results inthe incorporation of a modified, or non-naturally occurring base intothe target nucleic acid.

Mutations in a gene or pathway described herein may be corrected usingone of the approaches discussed herein. In an embodiment, a mutation ina gene or pathway described herein is corrected by homology directedrepair (HDR) using a template nucleic acid. In an embodiment, a mutationin a gene or pathway described herein is corrected by homologousrecombination (HR) using a template nucleic acid. In an embodiment, amutation in a gene or pathway described herein is corrected byNon-Homologous End Joining (NHEJ) repair using a template nucleic acid.In other embodiments, nucleic acid encoding molecules of interest may beinserted at or near a site modified by a CRISPR system of the presentinvention. In embodiments, the template nucleic acid comprisesregulatory elements, e.g., one or more promotors and/or enhancers,operably linked to the nucleic acid sequence encoding a molecule ofinterest, e.g., as described herein.

HDR or HR Repair and Template Nucleic Acids

As described herein, nuclease-induced homology directed repair (HDR) orhomologous recombination (HR) can be used to alter a target sequence andcorrect (e.g., repair or edit) a mutation in the genome. While notwishing to be bound by theory, it is believed that alteration of thetarget sequence occurs by repair based on a donor template or templatenucleic acid. For example, the donor template or the template nucleicacid provides for alteration of the target sequence. It is contemplatedthat a plasmid donor or linear double stranded template can be used as atemplate for homologous recombination. It is further contemplated that asingle stranded donor template can be used as a template for alterationof the target sequence by alternate methods of homology directed repair(e.g., single strand annealing) between the target sequence and thedonor template. Donor template-effected alteration of a target sequencemay depend on cleavage by a Cas9 molecule. Cleavage by Cas9 can comprisea double strand break, one single strand break, or two single strandbreaks.

In an embodiment, a mutation can be corrected by either a singledouble-strand break or two single strand breaks. In an embodiment, amutation can be corrected by providing a template and a CRISPR/Cas9system that creates (1) one double strand break, (2) two single strandbreaks, (3) two double stranded breaks with a break occurring on eachside of the target sequence, (4) one double stranded break and twosingle strand breaks with the double strand break and two single strandbreaks occurring on each side of the target sequence, (5) four singlestranded breaks with a pair of single stranded breaks occurring on eachside of the target sequence, or (6) one single strand break.

Double Strand Break Mediated Correction

In an embodiment, double strand cleavage is effected by a Cas9 moleculehaving cleavage activity associated with an HNH-like domain and cleavageactivity associated with a RuvC-like domain, e.g., an N-terminalRuvC-like domain, e.g., a wild type Cas9. Such embodiments require onlya single gRNA.

Single Strand Break Mediated Correction

In other embodiments, two single strand breaks, or nicks, are effectedby a Cas9 molecule having nickase activity, e.g., cleavage activityassociated with an HNH-like domain or cleavage activity associated withan N-terminal RuvC-like domain. Such embodiments require two gRNAs, onefor placement of each single strand break. In an embodiment, the Cas9molecule having nickase activity cleaves the strand to which the gRNAhybridizes, but not the strand that is complementary to the strand towhich the gRNA hybridizes. In an embodiment, the Cas9 molecule havingnickase activity does not cleave the strand to which the gRNAhybridizes, but rather cleaves the strand that is complementary to thestrand to which the gRNA hybridizes.

In an embodiment, the nickase has HNH activity, e.g., a Cas9 moleculehaving the RuvC activity inactivated, e.g., a Cas9 molecule having amutation at D10, e.g., the D10A mutation. D10A inactivates RuvC;therefore, the Cas9 nickase has (only) HN H activity and will cut on thestrand to which the gRNA hybridizes (e.g., the complementary strand,which does not have the NGG PAM on it). In other embodiments, a Cas9molecule having an H840, e.g., an H840A, mutation can be used as anickase. H840A inactivates HNH; therefore, the Cas9 nickase has (only)RuvC activity and cuts on the non-complementary strand (e.g., the strandthat has the NGG PAM and whose sequence is identical to the gRNA).

In an embodiment, in which a nickase and two gRNAs are used to positiontwo single strand nicks, one nick is on the + strand and one nick is onthe − strand of the target nucleic acid. The PAMs are outwardly facing.The gRNAs can be selected such that the gRNAs are separated by, fromabout 0-50, 0-100, or 0-200 nucleotides. In an embodiment, there is nooverlap between the target sequence that is complementary to thetargeting domains of the two gRNAs. In an embodiment, the gRNAs do notoverlap and are separated by as much as 50, 100, or 200 nucleotides. Inan embodiment, the use of two gRNAs can increase specificity, e.g., bydecreasing off-target binding (Ran el al., CELL 2013).

In an embodiment, a single nick can be used to induce HDR. It iscontemplated herein that a single nick can be used to increase the ratioof HDR, HR or NHEJ at a given cleavage site.

Placement of the Double Strand Break or a Single Strand Break Relativeto Target Position

The double strand break or single strand break in one of the strandsshould be sufficiently close to target position such that correctionoccurs. In an embodiment, the distance is not more than 50, 100, 200,300, 350 or 400 nucleotides. While not wishing to be bound by theory, itis believed that the break should be sufficiently close to targetposition such that the break is within the region that is subject toexonuclease-mediated removal during end resection. If the distancebetween the target position and a break is too great, the mutation maynot be included in the end resection and, therefore, may not becorrected, as donor sequence may only be used to correct sequence withinthe end resection region.

In an embodiment, in which a gRNA (unimolecular (or chimeric) or modulargRNA) and Cas9 nuclease induce a double strand break for the purpose ofinducing HDR- or HR-mediated correction, the cleavage site is between0-200 bp (e.g., 0 to 175, 0 to 150, 0 to 125, 0 to 100, 0 to 75, 0 to50, 0 to 25, 25 to 200, 25 to 175, 25 to 150, 25 to 125, 25 to 100, 25to 75, 25 to 50, 50 to 200, 50 to 175, 50 to 150, 50 to 125, 50 to 100,50 to 75, 75 to 200, 75 to 175, 75 to 150, 75 to 125, 75 to 100 bp) awayfrom the target position. In an embodiment, the cleavage site is between0-100 bp (e.g., 0 to 75, 0 to 50, 0 to 25, 25 to 100, 25 to 75, 25 to50, 50 to 100, 50 to 75 or 75 to 100 bp) away from the target position.

In an embodiment, in which two gRNAs (independently, unimolecular (orchimeric) or modular gRNA) complexing with Cas9 nickases induce twosingle strand breaks for the purpose of inducing HDR-mediatedcorrection, the closer nick is between 0-200 bp (e.g., 0 to 175, 0 to150, 0 to 125, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 25 to 200, 25 to175, 25 to 150, 25 to 125, 25 to 100, 25 to 75, 25 to 50, 50 to 200, 50to 175, 50 to 150, 50 to 125, 50 to 100, 50 to 75, 75 to 200, 75 to 175,75 to 150, 75 to 125, 75 to 100 bp) away from the target position andthe two nicks will ideally be within 25-55 bp of each other (e.g., 25 to50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 55, 30 to 50, 30 to45, 30 to 40, 30 to 35, 35 to 55, 35 to 50, 35 to 45, 35 to 40, 40 to55, 40 to 50, 40 to 45 bp) and no more than 100 bp away from each other(e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20, 10 or 5 bp away fromeach other). In an embodiment, the cleavage site is between 0-100 bp(e.g., 0 to 75, 0 to 50, 0 to 25, 25 to 100, 25 to 75, 25 to 50, 50 to100, 50 to 75 or 75 to 100 bp) away from the target position.

In one embodiment, two gRNAs, e.g., independently, unimolecular (orchimeric) or modular gRNA, are configured to position a double-strandbreak on both sides of a target position. In an alternate embodiment,three gRNAs, e.g., independently, unimolecular (or chimeric) or modulargRNA, are configured to position a double strand break (i.e., one gRNAcomplexes with a Cas9 nuclease) and two single strand breaks or pairedsingle stranded breaks (i.e., two gRNAs complex with Cas9 nickases) oneither side of the target position (e.g., the first gRNA is used totarget upstream (i.e., 5′) of the target position and the second gRNA isused to target downstream (i.e., 3′) of the target position). In anotherembodiment, four gRNAs, e.g., independently, unimolecular (or chimeric)or modular gRNA, are configured to generate two pairs of single strandedbreaks (i.e., two pairs of two gRNAs complex with Cas9 nickases) oneither side of the target position (e.g., the first gRNA is used totarget upstream (i.e., 5′) of the target position and the second gRNA isused to target downstream (i.e., 3′) of the target position). The doublestrand break(s) or the closer of the two single strand nicks in a pairwill ideally be within 0-500 bp of the target position (e.g., no morethan 450, 400, 350, 300, 250, 200, 150, 100, 50 or 25 bp from the targetposition). When nickases are used, the two nicks in a pair are within25-55 bp of each other (e.g., between 25 to 50, 25 to 45, 25 to 40, 25to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to50, 35. to 50, 40 to 50, 45 to 50, 35 to 45, or 40 to 45 bp) and no morethan 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50,40, 30, 20 or 10 bp).

In one embodiment, two gRNAs, e.g., independently, unimolecular (orchimeric) or modular gRNA, are configured to position a double-strandbreak on both sides of a target position. In an alternate embodiment,three gRNAs, e.g., independently, unimolecular (or chimeric) or modulargRNA, are configured to position a double strand break (i.e., one gRNAcomplexes with a Cas9 nuclease) and two single strand breaks or pairedsingle stranded breaks (i.e., two gRNAs complex with Cas9 nickases) ontwo target sequences (e.g., the first gRNA is used to target an upstream(i.e., 5′) target sequence and the second gRNA is used to target adownstream (i.e., 3′) target sequence of an insertion site. In anotherembodiment, four gRNAs, e.g., independently, unimolecular (or chimeric)or modular gRNA, are configured to generate two pairs of single strandedbreaks (i.e., two pairs of two gRNAs complex with Cas9 nickases) oneither side of an insertion site (e.g., the first gRNA is used to targetan upstream (i.e., 5′) target sequence described herein, and the secondgRNA is used to target a downstream (i.e., 3′) target sequence describedherein). The double strand break(s) or the closer of the two singlestrand nicks in a pair will ideally be within 0-500 bp of the targetposition (e.g., no more than 450, 400, 350, 300, 250, 200, 150, 100, 50or 25 bp from the target position). When nickases are used, the twonicks in a pair are within 25-55 bp of each other (e.g., between 25 to50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 35 to45, or 40 to 45 bp) and no more than 100 bp away from each other (e.g.,no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp).

Length of the Homology Arms

The homology arm should extend at least as far as the region in whichend resection may occur, e.g., in order to allow the resected singlestranded overhang to find a complementary region within the donortemplate. The overall length could be limited by parameters such asplasmid size or viral packaging limits. In an embodiment, a homology armdoes not extend into repeated elements, e.g., ALU repeats, LINE repeats.A template may have two homology arms of the same or different lengths.

Exemplary homology arm lengths include at least 25, 50, 100, 250, 500,750 or 1000 nucleotides.

Target position, as used herein, refers to a site on a target nucleicacid (e.g., the chromosome) that is modified by a Cas9molecule-dependent process. For example, the target position can be amodified Cas9 molecule cleavage of the target nucleic acid and templatenucleic acid directed modification, e.g., correction, of the targetposition. In an embodiment, a target position can be a site between twonucleotides, e.g., adjacent nucleotides, on the target nucleic acid intowhich one or more nucleotides is added. The target position may compriseone or more nucleotides that are altered, e.g., corrected, by a templatenucleic acid. In an embodiment, the target position is within a targetsequence (e.g., the sequence to which the gRN A binds) In an embodiment,a target position is upstream or downstream of a target sequence (e.g.,the sequence to which the gRNA binds).

Typically, the template sequence undergoes a breakage mediated orcatalyzed recombination with the target sequence. In an embodiment, thetemplate nucleic acid includes sequence that corresponds to a site onthe target sequence that is cleaved by a Cas9 mediated cleavage event.In an embodiment, the template nucleic acid includes sequence thatcorresponds to both, a first site on the target sequence that is cleavedin a first Cas9 mediated event, and a second site on the target sequencethat is cleaved in a second Cas9 mediated event.

In an embodiment, the template nucleic acid can include sequence whichresults in an alteration in the coding sequence of a translatedsequence, e.g., one which results in the substitution of one amino acidfor another in a protein product, e.g., transforming a mutant alleleinto a wild type allele, transforming a wild type allele into a mutantallele, and/or introducing a stop codon, insertion of an amino acidresidue, deletion of an amino acid residue, or a nonsense mutation.

In other embodiments, the template nucleic acid can include sequencewhich results in an alteration in a non-coding sequence, e.g., analteration in an exon or in a 5′ or 3′ non-translated or non-transcribedregion. Such alterations include an alteration in a control element,e.g., a promoter, enhancer, and an alteration in a cis-acting ortrans-acting control element.

The template nucleic acid can include sequence which, when integrated,results in:

decreasing the activity of a positive control element;

increasing the activity of a positive control element;

decreasing the activity of a negative control element;

increasing the activity of a negative control element;

decreasing the expression of a gene;

increasing the expression of a gene;

increasing resistance to a disorder or disease;

increasing resistance to viral entry;

correcting a mutation or altering an unwanted amino acid residue;

conferring, increasing, abolishing or decreasing a biological propertyof a gene product, e.g., increasing the enzymatic activity of an enzyme,or increasing the ability of a gene product to interact with anothermolecule.

The template nucleic acid can include sequence which results in:

a change in sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or morenucleotides of the target sequence.

In an embodiment, the template nucleic acid is 20+/−10, 30+/−10,40+/−10, 50+/−10, 60+/−10, 70+/−10, 80+/−10, 90+/−10, 100+/−10,110+/−10, 120+/−10, 130+/−10, 140+/−10, 150+/−10, 160+/−10, 170+/−10,180+/−10, 190+/−10, 200+/−10, 210+/−10, 220+/−10, 200-300, 300-400,400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-2000,2000-3000 or more than 3000 nucleotides in length.

A template nucleic acid comprises the following components:

[5′ homology arm]-[insertion sequence]-[3′ homology arm].

The homology arms provide for recombination into the chromosome, whichcan replace the undesired element, e.g., a mutation or signature, withthe replacement sequence. In an embodiment, the homology arms flank themost distal cleavage sites.

In an embodiment, the 3′ end of the 5′ homology arm is the position nextto the 5′ end of the replacement sequence. In an embodiment, the 5′homology arm can extend at least 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 120, 150, 180, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500,or 2000 nucleotides 5′ from the 5′ end of the replacement sequence.

In an embodiment, the 5′ end of the 3′ homology arm is the position nextto the 3′ end of the replacement sequence. In an embodiment, the 3′homology arm can extend at least 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 120, 150, 180, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500,or 2000 nucleotides 3′ from the 3′ end of the replacement sequence.

It is contemplated herein that one or both homology arms may beshortened to avoid including certain sequence repeat elements, e.g., Alurepeats, LINE elements. For example, a 5′ homology arm may be shortenedto avoid a sequence repeat element. In other embodiments, a 3′ homologyarm may be shortened to avoid a sequence repeat element. In someembodiments, both the 5′ and the 3′ homology arms may be shortened toavoid including certain sequence repeat elements.

It is contemplated herein that template nucleic acids for correcting amutation may designed for use as a single-stranded oligonucleotide(ssODN). When using a ssODN, 5′ and 3′ homology arms may range up toabout 200 base pairs (bp) in length, e.g., at least 25, 50, 75, 100,125, 150, 175, or 200 bp in length. Longer homology arms are alsocontemplated for ssODNs as improvements in oligonucleotide synthesiscontinue to be made.

NHEJ Approaches for Gene Targeting.

As described herein, nuclease-induced non-homologous end joining (NHEJ)can be used to target gene-specific knockouts. Nuclease-induced NHEJ canalso be used to remove (e.g., delete) sequence in a gene of interest.

While not wishing to be bound by theory, it is believed that, in anembodiment, the genomic alterations associated with the methodsdescribed herein rely on nuclease-induced NHEJ and the error-pronenature of the NHEJ repair pathway. NHEJ repairs a double-strand break inthe DNA by joining together the two ends; however, generally, theoriginal sequence is restored only if two compatible ends, exactly asthey were formed by the double-strand break, are perfectly ligated. TheDNA ends of the double-strand break are frequently the subject ofenzymatic processing, resulting in the addition or removal ofnucleotides, at one or both strands, prior to rejoining of the ends.This results in the presence of insertion and/or deletion (indel)mutations in the DNA sequence at the site of the NHEJ repair. Two-thirdsof these mutations may alter the reading frame and, therefore, produce anon-functional protein. Additionally, mutations that maintain thereading frame, but which insert or delete a significant amount ofsequence, can destroy functionality of the protein. This is locusdependent as mutations in critical functional domains are likely lesstolerable than mutations in non-critical regions of the protein.

The indel mutations generated by NHEJ are unpredictable in nature;however, at a given break site certain indel sequences are favored andare over represented in the population. The lengths of deletions canvary widely; most commonly in the 1-50 bp range, but they can easilyreach greater than 100-200 bp. Insertions tend to be shorter and ofteninclude short duplications of the sequence immediately surrounding thebreak site. However, it is possible to obtain large insertions, and inthese cases, the inserted sequence has often been traced to otherregions of the genome or to plasmid DNA present in the cells.

Because NHEJ is a mutagenic process, it can also be used to delete smallsequence motifs as long as the generation of a specific final sequenceis not required. If a double-strand break is targeted near to a shorttarget sequence, the deletion mutations caused by the NHEJ repair oftenspan, and therefore remove, the unwanted nucleotides. For the deletionof larger DNA segments, introducing two double-strand breaks, one oneach side of the sequence, can result in NHEJ between the ends withremoval of the entire intervening sequence. Both of these approaches canbe used to delete specific DNA sequences; however, the error-pronenature of NHEJ may still produce indel mutations at the site of repair.

Both double strand cleaving Cas9 molecules and single strand, ornickase, Cas9 molecules can be used in the methods and compositionsdescribed herein to generate NHEJ-mediated indels. NHEJ-mediated indelstargeted to the gene, e.g., a coding region, e.g., an early codingregion of a gene of interest can be used to knockout (i.e., eliminateexpression of) a gene of interest. For example, early coding region of agene of interest includes sequence immediately following a transcriptionstart site, within a first exon of the coding sequence, or within 500 bpof the transcription start site (e.g., less than 500, 450, 400, 350,300, 250, 200, 150, 100 or 50 bp).

Placement of Double Strand or Single Strand Breaks Relative to theTarget Position

In an embodiment, in which a gRNA and Cas9 nuclease generate a doublestrand break for the purpose of inducing NHEJ-mediated indels, a gRNA,e.g., a unimolecular (or chimeric) or modular gRNA molecule, isconfigured to position one double-strand break in close proximity to anucleotide of the target position. In an embodiment, the cleavage siteis between 0-500 bp away from the target position (e.g., less than 500,400, 300, 200, 100, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2or 1 bp from the target position).

In an embodiment, in which two gRNAs complexing with Cas9 nickasesinduce two single strand breaks for the purpose of inducingNHEJ-mediated indels, two gRNAs, e.g., independently, unimolecular (orchimeric) or modular gRNA, are configured to position two single-strandbreaks to provide for NHEJ repair a nucleotide of the target position.In an embodiment, the gRNAs are configured to position cuts at the sameposition, or within a few nucleotides of one another, on differentstrands, essentially mimicking a double strand break. In an embodiment,the closer nick is between 0-30 bp away from the target position (e.g.,less than 30, 25, 20, 1, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 bp from thetarget position), and the two nicks are within 25-55 bp of each other(e.g., between 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to50, 45 to 50, 35 to 45, or 40 to 45 bp) and no more than 100 bp awayfrom each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10bp). In an embodiment, the gRNAs are configured to place a single strandbreak on either side of a nucleotide of the target position.

Both double strand cleaving Cas9 molecules and single strand, ornickase, Cas9 molecules can be used in the methods and compositionsdescribed herein to generate breaks both sides of a target position.Double strand or paired single strand breaks may be generated on bothsides of a target position to remove the nucleic acid sequence betweenthe two cuts (e.g., the region between the two breaks is deleted). Inone embodiment, two gRNAs, e.g., independently, unimolecular (orchimeric) or modular gRNA, are configured to position a double-strandbreak on both sides of a target position (e.g., the first gRNA is usedto target upstream (i.e., 5′) of the mutation in a gene or pathwaydescribed herein, and the second gRNA is used to target downstream(i.e., 3′) of the mutation in a gene or pathway described herein). In analternate embodiment, three gRNAs, e.g., independently, unimolecular (orchimeric) or modular gRNA, are configured to position a double strandbreak (i.e., one gRNA complexes with a Cas9 nuclease) and two singlestrand breaks or paired single stranded breaks (i.e., two gRNAs complexwith Cas9 nickases) on either side of a target position (e.g., the firstgRNA is used to target upstream (i.e., 5′) of the mutation in a gene orpathway described herein, and the second gRNA is used to targetdownstream (i.e., 3′) of the mutation in a gene or pathway describedherein). In another embodiment, four gRNAs, e.g., independently,unimolecular (or chimeric) or modular gRNA, are configured to generatetwo pairs of single stranded breaks (i.e., two pairs of two gRNAscomplex with Cas9 nickases) on either side of the target position (e.g.,the first gRNA is used to target upstream (i.e., 5′) of the mutation ina gene or pathway described herein, and the second gRNA is used totarget downstream (i.e., 3′) of the mutation in a gene or pathwaydescribed herein). The double strand break(s) or the closer of the twosingle strand nicks in a pair will ideally be within 0-500 bp of thetarget position (e.g., no more than 450, 400, 350, 300, 250, 200, 150,100, 50 or 25 bp from the target position). When nickases are used, thetwo nicks in a pair are within 25-55 bp of each other (e.g., between 25to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 35 to45, or 40 to 45 bp) and no more than 100 bp away from each other (e.g.,no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp).

In other embodiments, the insertion of template nucleic acid may bemediated by microhomology end joining (MMEJ). See, e.g., Saksuma et al.,“MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITChsystems.” Nature Protocols 11, 118-133 (2016) doi:10.1038/nprot.2015.140Published online 17 Dec. 2015, the contents of which are incorporated byreference in their entirety.

VIII. Systems Comprising More than One gRNA Molecule

While not intending to be bound by theory, it has been surprisinglyshown herein that the targeting of two target sequences (e.g., by twogRNA molecule/Cas9 molecule complexes which each induce a single- ordouble-strand break at or near their respective target sequences)located in close proximity on a continuous nucleic acid induces excision(e.g., deletion) of the nucleic acid sequence (or at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% of the nucleic acid sequence) locatedbetween the two target sequences. In some aspects, the presentdisclosure provides for the use of two or more gRNA molecules thatcomprise targeting domains targeting target sequences in close proximityon a continuous nucleic acid, e.g., a chromosome, e.g., a gene or genelocus, including its introns, exons and regulatory elements. The use maybe, for example, by introduction of the two or more gRNA molecules,together with one or more Cas9 molecules (or nucleic acid encoding thetwo or more gRNA molecules and/or the one or more Cas9 molecules) into acell.

In some aspects, the target sequences of the two or more gRNA moleculesare located at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000,5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, or15,000 nucleotides apart on a continuous nucleic acid, but not more than25,000 nucleotides apart on a continuous nucleic acid. In embodiments,the target sequences are located between about 4000 and about 6000nucleotides apart. In an embodiment, the target sequences are locatedabout 4000 nucleotides apart. In an embodiment, the target sequences arelocated about 5000 nucleotides apart. In an embodiment, the targetsequences are located about 6000 nucleotides apart.

In some aspects, the plurality of gRNA molecules each target sequenceswithin the same gene or gene locus. In an exemplary aspect, the targetsequences of the two or more gRNA molecules are located in the HBG1promoter region. In an exemplary aspect, the target sequences of the twoor more gRNA molecules are located in the HBG2 promoter region. Inanother aspect, the plurality of gRNA molecules each target sequenceswithin 2 or more different genes or gene loci. In an exemplary aspect,the target sequence of one or more of the gRNA molecules is located inthe HBG1 promoter region, and the target sequence of one or more of theother gRNA molecules is located in the HBG2 promoter region.

In some aspects, the invention provides compositions and cellscomprising a plurality, for example, 2 or more, for example, 2, gRNAmolecules of the invention, wherein the plurality of gRNA moleculestarget sequences less than 15,000, less than 14,000, less than 13,000,less than 12,000, less than 11,000, less than 10,000, less than 9,000,less than 8,000, less than 7,000, less than 6,000, less than 5,000, lessthan 4,000, less than 3,000, less than 2,000, less than 1,000, less than900, less than 800, less than 700, less than 600, less than 500, lessthan 400, less than 300, less than 200, less than 100, less than 90,less than 80, less than 70, less than 60, less than 50, less than 40, orless than 30 nucleotides apart. In an embodiment, the target sequencesare on the same strand of duplex nucleic acid. In an embodiment, thetarget sequences are on different strands of duplex nucleic acid.

In one embodiment, the invention provides a method for excising (e.g.,deleting) nucleic acid disposed between two gRNA binding sites disposedless than 25,000, less than 20,000, less than 15,000, less than 14,000,less than 13,000, less than 12,000, less than 11,000, less than 10,000,less than 9,000, less than 8,000, less than 7,000, less than 6,000, lessthan 5,000, less than 4,000, less than 3,000, less than 2,000, less than1,000, less than 900, less than 800, less than 700, less than 600, lessthan 500, less than 400, less than 300, less than 200, less than 100,less than 90, less than 80, less than 70, less than 60, less than 50,less than 40, or less than 30 nucleotides apart on the same or differentstrands of duplex nucleic acid. In an embodiment, the method providesfor deletion of more than 50%, more than 60%, more than 70%, more than80%, more than 85%, more than 86%, more than 87%, more than 88%, morethan 89%, more than 90%, more than 91%, more than 92%, more than 93%,more than 94%, more than 95%, more than 96%, more than 97%, more than98%, more than 99%, or 100% of the nucleotides disposed between the PAMsites associated with each gRNA binding site. In embodiments, thedeletion further comprises of one or more nucleotides within one or moreof the PAM sites associated with each gRNA binding site. In embodiments,the deletion also comprises one or more nucleotides outside of theregion between the PAM sites associated with each gRNA binding site.

In one aspect, the two or more gRNA molecules comprise targeting domainstargeting target sequences flanking a gene regulatory element, e.g., apromotor binding site, an enhancer region, or a repressor region, suchthat excision of the intervening sequence (or a portion of theintervening sequence) causes up- or down-regulation of a gene ofinterest. In other embodiments, the two or more gRNA molecules comprisetargeting domains that target sequences flanking a gene, such thatexcision of the intervening sequence (or portion thereof) causesdeletion of the gene of interest.

In an embodiment, the two or more gRNA molecules each include atargeting domain comprising, e.g., consisting of, a targeting domainsequence of Table 1, e.g., of Table 2 or, e.g., of Table 3a or Table 3b.In embodiments, the two or more gRNA molecules each include a targetingdomain comprising, e.g., consisting of, the targeting domain of a gRNAmolecule which results in at least 15% upregulation in the number of Fcells in a population of red blood cells differentiated (e.g., at day 7following editing) from HSPCs edited by said gRNA ex vivo by the methodsdescribed herein. In aspects, the two or more gRNA molecules comprisetargeting domains that are complementary with sequences in the same geneor region, e.g., the HBG1 promoter region or the HBG2 promoter region.In aspects, the two or more gRNA molecules comprise targeting domainsthat are complementary with sequences of different genes or regions, forexample one in the HBG1 promoter region and one in the HBG2 promoterregion.

In one aspect, the two or more gRNA molecules comprise targeting domainstargeting target sequences flanking a gene regulatory element, e.g., apromotor binding site, an enhancer region, or a repressor region, suchthat excision of the intervening sequence (or a portion of theintervening sequence) causes up- or down-regulation of a gene ofinterest. In another aspect, the two or more gRNA molecules comprisetargeting domains targeting target sequences flanking a gene, such thatexcision of the intervening sequence (or a portion of the interveningsequence) causes deletion of the gene of interest. By way of example,the two or more gRNA molecules comprise targeting domains targetingtarget sequences flanking the HBG1 gene (for example, one gRNA moleculetargeting a target sequence in the HBG1 promoter region, and a secondgRNA molecule targeting a target sequence in the HBG2 promoter region),such that the HBG1 gene is excised.

In an embodiment, the two or more gRNA molecules comprise targetingdomains that comprise, e.g., consist of, targeting domains selected fromTable 1.

In aspects, the two or more gRNA molecules comprise targeting domainscomprising, e.g., consisting of, targeting domain sequences listed inTable 2, above. In aspects, the two or more gRNA molecules comprisetargeting domains comprising, e.g., consisting of, targeting domainsequences of gRNAs listed in Table 3a, above. In aspects, the two ormore gRNA molecules comprise targeting domains comprising, e.g.,consisting of, targeting domain sequences listed in Table 3b, above.

The gRNA molecules comprising targeting domains which target sequencesof within a nondeletional HPFH region, e.g., an HBG1 and/or HBG2promoter region, may additionally be used with a gRNA moleculecomprising a targeting domain complementary to a target sequence within,for example, a BCL11a enhancer region (e.g., a +55, +58 or +62 BCL11aenhancer region) and/or a gRNA molecule comprising a targeting domaincomplementary to a target sequence of a deletional HPFH locus. Suchdeletional HPFH loci are known in the art, for example, those describedin Sankaran V G et al. NEJM (2011) 365:807-814 (hereby incorporated byreference in its entirety).

IX. Properties of the gRNA

It has further been surprisingly shown herein that single gRNA moleculesmay have target sequences in more than one loci (for example, loci withhigh sequence homology), and that, when such loci are present on thesame chromosome, for example, within less than about 15,000 nucleotides,less than about 14,000 nucleotides, less than about 13,000 nucleotides,less than about 12,000 nucleotides, less than about 11,000 nucleotides,less than about 10,000 nucleotides, less than about 9,000 nucleotides,less than about 8,000 nucleotides, less than about 7,000 nucleotides,less than about 6,000 nucleotides, less than about 5,000 nucleotides,less than about 4,000 nucleotides, or less than about 3,000 nucleotides,(e.g., from about 4,000 to about 6,000 nucleotides apart) such a gRNAmolecule may result in excision of the intervening sequence (or portionthereof), thereby resulting in a beneficial effect, for example,upregulation of fetal hemoglobin in erythroid cells differentiated frommodified HSPCs (as described herein). Thus, in an aspect, the inventionprovides gRNA molecules which have target sequences at two loci, forexample, to loci on the same chromosome, for example, which have targetsequences at an HBG1 promoter region and at an HBG2 promoter region (forexample as described in Table 1). Without begin bound by theory, it isbelieved that such gRNAs may result in the cutting of the genome at morethan one location (e.g., at the target sequence in each of two regions),and that subsequent repair may result in a deletion of the interveningnucleic acid sequence. Again, without being bound by theory, deletion ofsaid intervening sequence may have a desired effect on the expression orfunction of one or more proteins.

It has further been surprisingly shown that gRNA molecules whichcomprise a targeting domain complementary to a sequence only in one geneor region, for example, which is complementary to a target sequence inthe HBG1 promoter region (but not the HBG2 promoter region), or which iscomplementary to a target sequence in the HBG2 promoter region (but notthe HBG1 promoter region), can result in significant upregulation offetal hemoglobin in erythroid cells differentiated from modified HSPCs(as described herein). In aspects, the invention thus provides gRNAmolecules which comprise a targeting domain which is complementary to atarget sequence in a single nondeletional HPFH region, for examplewithin a HBG1 or HBG2 promoter region (for example as described in Table1), but which does not have a fully (e.g., 100%) complementary targetsequence match in any other gene or region.

It has further been surprisingly shown herein that gRNA molecules andCRISPR systems comprising said gRNA molecules produce similar oridentical indel patterns across multiple experiments using the same celltype, method of delivery and crRNA/tracr components. Without being boundby theory, it is believed that some indel patterns may be moreadvantageous than others. For example, indels which predominantlyinclude insertions and/or deletions which result in a “frameshiftmutation” (e.g., 1- or 2-base pair insertion or deletions, or anyinsertion or deletion where n/3 is not a whole number (where n=thenumber of nucleotides in the insertion or deletion)) may be beneficialin reducing or eliminating expression of a functional protein. Likewise,indels which predominantly include “large deletions” (deletions of morethan 10, 11, 12, 13, 14, 15, 20, 25, or 30 nucleotides, for example,more than 1 kb, more than 2 kb, more than 3 kb, more than 5 kb or morethan 10 kb, for example, comprising sequence disposed between a firstand second binding site for a gRNA, e.g., as described herein) may alsobe beneficial in, for example, removing critical regulatory sequencessuch as promoter binding sites, or altering the structure or function ofa locus, which may similarly have an effect on expression of functionalprotein. While the indel patterns induced by a given gRNA/CRISPR systemhave surprisingly been found to be consistently reproduced for a givencell type, gRNA and CRISPR system, as described herein, not any singleindel structure will inevitably be produced in a given cell uponintroduction of a gRNA/CRISPR system.

The invention thus provides for gRNA molecules which create a beneficialindel pattern or structure, for example, which have indel patterns orstructures predominantly composed of large deletions. Such gRNAmolecules may be selected by assessing the indel pattern or structurecreated by a candidate gRNA molecule in a test cell (for example, aHEK293 cell) or in the cell of interest, e.g., a HSPC cell by NGS, asdescribed herein. As shown in the Examples, gRNA molecules have beendiscovered, which, when introduced into the desired cell population,result in a population of cells comprising a significant fraction of thecells having a large deletion at or near the target sequence of thegRNA. In some cases, the rate of large deletion indel formation is ashigh as 75%, 80%, 85%, 90% or more. The invention thus provides forpopulations of cells which comprise at least about 40% of cells (e.g.,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or at least about 99%) having a large deletion, e.g., asdescribed herein, at or near the target site of a gRNA moleculedescribed herein. The invention also provides for populations of cellswhich comprise at least about 50% of cells (e.g., at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, or at least about 99%) having a large deletion, e.g.,as described herein, at or near the target site of a gRNA moleculedescribed herein.

The invention thus provides methods of selecting gRNA molecules for usein the therapeutic methods of the invention comprising: 1) providing aplurality of gRNA molecules to a target of interest, 2) assessing theindel pattern or structure created by use of said gRNA molecules, 3)selecting a gRNA molecule that forms an indel pattern or structurecomposed predominantly of frameshift mutations, large deletions or acombination thereof, and 4) using said selected gRNA in a methods of theinvention.

The invention thus provides methods of selecting gRNA molecules for usein the therapeutic methods of the invention comprising: 1) providing aplurality of gRNA molecules to a target of interest, e.g., which havetarget sequences at more than one location 2) assessing the indelpattern or structure created by use of said gRNA molecules, 3) selectinga gRNA molecule that forms an excision of sequence comprising nucleicacid sequence located between the two target sequences, e.g., in atleast about 25% or more of the cells of a population of cells which areexposed to said gRNA molecules, and 4) using said selected gRNA moleculein a methods of the invention.

The invention further provides methods of altering cells, and alteredcells, wherein a particular indel pattern is constantly produced with agiven gRNA/CRISPR system in that cell type. The indel patterns,including the top 5 most frequently occurring indels observed with thegRNA/CRISPR systems described herein can be determined using the methodsof the examples, and are disclosed, for example, in the Examples. Asshown in the Examples, populations of cells are generated, wherein asignificant fraction of the cells comprises one of the top 5 indels (forexample, populations of cells wherein one of the top 5 indels is presentin more than 30%, more than 40%, more than 50%, more than 60% or more ofthe cells of the population. Thus, the invention provides cells, e.g.,HSPCs (as described herein), which comprise an indel of any one of thetop 5 indels observed with a given gRNA/CRISPR system. Further, theinvention provides populations of cells, e.g., HSPCs (as describedherein), which when assessed by, for example, NGS, comprise a highpercentage of cells comprising one of the top 5 indels described hereinfor a given gRNA/CRISPR system. When used in connection with indelpattern analysis, a “high percentage” refers to at least about 50%(e.g., at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, or at least about 99%) ofthe cells of the population comprising one of the top 5 indels describedherein for a given gRNA/CRISPR system. In other embodiments, thepopulation of cells comprises at least about 25% (e.g., from about 25%to about 60%, e.g., from about 25% to about 50%, e.g., from about 25% toabout 40%, e.g., from about 25% to about 35%) of cells which have one ofthe top 5 indels described herein for a given gRNA/CRISPR system.

It has also been discovered that certain gRNA molecules do not createindels at off-target sequences (e.g., off-target sequences outside ofthe HBG1 and/or HBG2 promoter region) within the genome of the targetcell type, or produce indels at off target sites (e.g., off-targetsequences outside of the HBG1 and/or HBG2 promoter region) at very lowfrequencies (e.g., <5% of cells within a population) relative to thefrequency of indel creation at the target site. Thus, the inventionprovides for gRNA molecules and CRISPR systems which do not exhibitoff-target indel formation in the target cell type, or which produce afrequency of off-target indel formation of less than 5%, for example, anindel at any off-target site outside of the HBG1 and/or HBG2 promoterregion at a frequency of less than 5%. In embodiments, the inventionprovides gRNA molecules and CRISPR systems which do not exhibit any offtarget indel formation in the target cell type. Thus, the inventionfurther provides a cell, e.g., a population of cells, e.g., HSPCs, e.g.,as described herein, which comprise an indel at or near a target site ofa gRNA molecule described herein (e.g., a frameshift indel, or any oneof the top 5 indels produced by a given gRNA/CRISPR system, e.g., asdescribed herein), but does not comprise an indel at any off-target siteof the gRNA molecule, for example, an indel at any off-target siteoutside of the HBG1 and/or HBG2 promoter region. In other embodiments,the invention further provides a population of cells, e.g., HSPCs, e.g.,as described herein, which comprises at least 20%, for example at least30%, for example at least 40%, for example at least 50%, for example atleast 60%, for example at least 70%, for example at least 75% of cellswhich have an indel at or near a target site of a gRNA moleculedescribed herein (e.g., a frameshift indel, or any one of the top 5indels produced by a given gRNA/CRISPR system, e.g., as describedherein), but which comprises less than 5%, e.g., less than 4%, less than3%, less than 2% or less than 1%, of cells comprising an indel at anyoff-target site of the gRNA molecule, for example, an indel at anyoff-target site outside of the HBG1 and/or HBG2 promoter region. Inother embodiments, the invention further provides a population of cells,e.g., HSPCs, e.g., as described herein, which comprises at least 20%,for example at least 30%, for example at least 40%, for example at least50%, for example at least 60%, for example at least 70%, for example atleast 75%, for example at least 80%, for example at least 90%, forexample at least 95%, of cells which have an indel within the HBG1and/or HBG2 promoter region (e.g., at or near a sequence which is asleast 90% homologous to the target sequence of the gRNA), but whichcomprises less than 5%, e.g., less than 4%, less than 3%, less than 2%or less than 1%, of cells comprising an indel at or near any off-targetsite outside of the HBG1 and/or HBG2 promoter region.

In embodiments, the off-target indel is formed within a sequence of agene, e.g., within a coding sequence of a gene. In embodiments nooff-target indel is formed within a sequence of a gene, e.g., within acoding sequence of a gene, in the cell of interest, e.g., as describedherein.

X. Delivery/Constructs

The components, e.g., a Cas9 molecule or gRNA molecule, or both, can bedelivered, formulated, or administered in a variety of forms. As anon-limiting example, the gRNA molecule and Cas9 molecule can beformulated (in one or more compositions), directly delivered oradministered to a cell in which a genome editing event is desired.Alternatively, nucleic acid encoding one or more components, e.g., aCas9 molecule or gRNA molecule, or both, can be formulated (in one ormore compositions), delivered or administered. In one aspect, the gRNAmolecule is provided as DNA encoding the gRNA molecule and the Cas9molecule is provided as DNA encoding the Cas9 molecule. In oneembodiment, the gRNA molecule and Cas9 molecule are encoded on separatenucleic acid molecules. In one embodiment, the gRNA molecule and Cas9molecule are encoded on the same nucleic acid molecule. In one aspect,the gRNA molecule is provided as RNA and the Cas9 molecule is providedas DNA encoding the Cas9 molecule. In one embodiment, the gRNA moleculeis provided with one or more modifications, e.g., as described herein.In one aspect, the gRNA molecule is provided as RNA and the Cas9molecule is provided as mRNA encoding the Cas9 molecule. In one aspect,the gRNA molecule is provided as RNA and the Cas9 molecule is providedas a protein. In one embodiment, the gRNA and Cas9 molecule are providedas a ribonuclear protein complex (RNP). In one aspect, the gRNA moleculeis provided as DNA encoding the gRNA molecule and the Cas9 molecule isprovided as a protein.

Delivery, e.g., delivery of the RNP, (e.g., to HSPC cells as describedherein) may be accomplished by, for example, electroporation (e.g., asknown in the art) or other method that renders the cell membranepermeable to nucleic acid and/or polypeptide molecules. In embodiments,the CRISPR system, e.g., the RNP as described herein, is delivered byelectroporation using a 4D-Nucleofector (Lonza), for example, usingprogram CM-137 on the 4D-Nucleofector (Lonza). In embodiments, theCRISPR system, e.g., the RNP as described herein, is delivered byelectroporation using a voltage from about 800 volts to about 2000volts, e.g., from about 1000 volts to about 1800 volts, e.g., from about1200 volts to about 1800 volts, e.g., from about 1400 volts to about1800 volts, e.g., from about 1600 volts to about 1800 volts, e.g., about1700 volts, e.g., at a voltage of 1700 volts. In embodiments, the pulsewidth/length is from about 10 ms to about 50 ms, e.g., from about 10 msto about 40 ms, e.g., from about 10 ms to about 30 ms, e.g., from about15 ms to about 25 ms, e.g., about 20 ms, e.g., 20 ms. In embodiments, 1,2, 3, 4, 5, or more, e.g., 2, e.g., 1 pulses are used. In an embodiment,the CRISPR system, e.g., the RNP as described herein, is delivered byelectroporation using a voltage of about 1700 volts (e.g., 1700 volts),a pulse width of about 20 ms (e.g., 20 ms), using a single (1) pulse. Inembodiments, electroporation is accomplished using a Neonelectroporator. Additional techniques for rendering the membranepermeable are known in the art and include, for example, cell squeezing(e.g., as described in WO2015/023982 and WO2013/059343, the contents ofwhich are hereby incorporated by reference in their entirety),nanoneedles (e.g., as described in Chiappini et al., Nat. Mat., 14;532-39, or US2014/0295558, the contents of which are hereby incorporatedby reference in their entirety) and nanostraws (e.g., as described inXie, ACS Nano, 7(5); 4351-58, the contents of which are herebyincorporated by reference in their entirety).

When a component is delivered encoded in DNA the DNA will typicallyinclude a control region, e.g., comprising a promoter, to effectexpression. Useful promoters for Cas9 molecule sequences include CMV,EF-1alpha, MSCV, PGK, CAG control promoters. Useful promoters for gRNAsinclude H1, EF-1a and U6 promoters. Promoters with similar or dissimilarstrengths can be selected to tune the expression of components.Sequences encoding a Cas9 molecule can comprise a nuclear localizationsignal (NLS), e.g., an SV40 NLS. In an embodiment, a promoter for a Cas9molecule or a gRNA molecule can be, independently, inducible, tissuespecific, or cell specific.

DNA-Based Delivery of a Cas9 Molecule and or a gRNA Molecule

DNA encoding Cas9 molecules and/or gRNA molecules, can be administeredto subjects or delivered into cells by art-known methods or as describedherein. For example, Cas9-encoding and/or gRNA-encoding DNA can bedelivered, e.g., by vectors (e.g., viral or non-viral vectors),non-vector based methods (e.g., using naked DNA or DNA complexes), or acombination thereof.

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered bya vector (e.g., viral vector/virus, plasmid, minicircle or nanoplasmid).

A vector can comprise a sequence that encodes a Cas9 molecule and/or agRNA molecule. A vector can also comprise a sequence encoding a signalpeptide (e.g., for nuclear localization, nucleolar localization,mitochondrial localization), fused, e.g., to a Cas9 molecule sequence.For example, a vector can comprise one or more nuclear localizationsequence (e.g., from SV40) fused to the sequence encoding the Cas9molecule.

One or more regulatory/control elements, e.g., a promoter, an enhancer,an intron, a polyadenylation signal, a Kozak consensus sequence,internal ribosome entry sites (IRES), a 2A sequence, and a spliceacceptor or donor can be included in the vectors. In some embodiments,the promoter is recognized by RNA polymerase II (e.g., a CMV promoter).In other embodiments, the promoter is recognized by RNA polymerase III(e.g., a U6 promoter). In some embodiments, the promoter is a regulatedpromoter (e.g., inducible promoter). In other embodiments, the promoteris a constitutive promoter. In some embodiments, the promoter is atissue specific promoter. In some embodiments, the promoter is a viralpromoter. In other embodiments, the promoter is a non-viral promoter.

In some embodiments, the vector or delivery vehicle is a minicircle. Insome embodiments, the vector or delivery vehicle is a nanoplasmid.

In some embodiments, the vector or delivery vehicle is a viral vector(e.g., for generation of recombinant viruses). In some embodiments, thevirus is a DNA virus (e.g., dsDNA or ssDNA virus). In other embodiments,the virus is an RNA virus (e.g., an ssRNA virus).

Exemplary viral vectors/viruses include, e.g., retroviruses,lentiviruses, adenovirus, adeno-associated virus (AAV), vacciniaviruses, poxviruses, and herpes simplex viruses. Viral vector technologyis well known in the art and is described, for example, in Sambrook etal., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, ColdSpring Harbor Press, NY), and in other virology and molecular biologymanuals.

In some embodiments, the virus infects dividing cells. In otherembodiments, the virus infects non-dividing cells. In some embodiments,the virus infects both dividing and non-dividing cells. In someembodiments, the virus can integrate into the host genome. In someembodiments, the virus is engineered to have reduced immunity, e.g., inhuman. In some embodiments, the virus is replication-competent. In otherembodiments, the virus is replication-defective, e.g., having one ormore coding regions for the genes necessary for additional rounds ofvirion replication and/or packaging replaced with other genes ordeleted. In some embodiments, the virus causes transient expression ofthe Cas9 molecule and/or the gRNA molecule. In other embodiments, thevirus causes long-lasting, e.g., at least 1 week, 2 weeks, 1 month, 2months, 3 months, 6 months, 9 months, 1 year, 2 years, or permanentexpression, of the Cas9 molecule and/or the gRNA molecule. The packagingcapacity of the viruses may vary, e.g., from at least about 4 kb to atleast about 30 kb, e.g., at least about 5 kb, 10 kb, 15 kb, 20 kb, 25kb, 30 kb, 35 kb, 40 kb, 45 kb, or 50 kb.

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered bya recombinant retrovirus. In some embodiments, the retrovirus (e.g.,Moloney murine leukemia virus) comprises a reverse transcriptase, e.g.,that allows integration into the host genome. In some embodiments, theretrovirus is replication-competent. In other embodiments, theretrovirus is replication-defective, e.g., having one of more codingregions for the genes necessary for additional rounds of virionreplication and packaging replaced with other genes, or deleted.

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered bya recombinant lentivirus. For example, the lentivirus isreplication-defective, e.g., does not comprise one or more genesrequired for viral replication.

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered bya recombinant adenovirus. In some embodiments, the adenovirus isengineered to have reduced immunity in human.

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered bya recombinant AAV. In some embodiments, the AAV can incorporate itsgenome into that of a host cell, e.g., a target cell as describedherein. In some embodiments, the AAV is a self-complementaryadeno-associated virus (scAAV), e.g., a scAAV that packages both strandswhich anneal together to form double stranded DNA. AAV serotypes thatmay be used in the disclosed methods include, e.g., AAV1, AAV2, modifiedAAV2 (e.g., modifications at Y444F, Y500F, Y730F and/or S662V), AAV3,modified AAV3 (e.g., modifications at Y705F, Y73 1 F and/or. T492V),AAV4, AAV5, AAV6, modified AAV6 (e.g., modifications at S663V and/orT492V), AAV8. AAV 8.2, AAV9, AAV rh 10, and pseudotyped AAV, such asAAV2/8, AAV2/5 and AAV2/6 can also be used in the disclosed methods.

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered bya hybrid virus, e.g., a hybrid of one or more of the viruses describedherein.

A Packaging cell is used to form a virus particle that is capable ofinfecting a host or target cell. Such a cell includes a 293 cell, whichcan package adenovirus, and a ψ2 cell or a PA317 cell, which can packageretrovirus. A viral vector used in gene therapy is usually generated bya producer cell line that packages a nucleic acid vector into a viralparticle. The vector typically contains the minimal viral sequencesrequired for packaging and subsequent integration into a host or targetcell (if applicable), with other viral sequences being replaced by anexpression cassette encoding the protein to be expressed. For example,an AAV vector used in gene therapy typically only possesses invertedterminal repeat (ITR) sequences from the AAV genome which are requiredfor packaging and gene expression in the host or target cell. Themissing viral functions are supplied in trans by the packaging cellline. Henceforth, the viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In an embodiment, the viral vector has the ability of cell type and/ortissue type recognition. For example, the viral vector can bepseudotyped with a different/alternative viral envelope glycoprotein;engineered with a cell type-specific receptor (e.g., geneticmodification of the viral envelope glycoproteins to incorporatetargeting ligands such as a peptide ligand, a single chain antibodies, agrowth factor); and/or engineered to have a molecular bridge with dualspecificities with one end recognizing a viral glycoprotein and theother end recognizing a moiety of the target cell surface (e.g.,ligand-receptor, monoclonal antibody, avidin-biotin and chemicalconjugation).

In an embodiment, the viral vector achieves cell type specificexpression. For example, a tissue-specific promoter can be constructedto restrict expression of the transgene (Cas 9 and gRNA) in only thetarget cell. The specificity of the vector can also be mediated bymicroRNA-dependent control of transgene expression. In an embodiment,the viral vector has increased efficiency of fusion of the viral vectorand a target cell membrane. For example, a fusion protein such asfusion-competent hemaglutinin (HA) can be incorporated to increase viraluptake into cells. In an embodiment, the viral vector has the ability ofnuclear localization. For example, a virus that requires the breakdownof the cell wall (during cell division) and therefore will not infect anon-diving cell can be altered to incorporate a nuclear localizationpeptide in the matrix protein of the virus thereby enabling thetransduction of non-proliferating cells.

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered bya non-vector based method (e.g., using naked DNA or DNA complexes). Forexample, the DNA can be delivered, e.g., by organically modified silicaor silicate (Ormosil), electroporation, gene gun, sonoporation,magnetofection, lipid-mediated transfection, dendrimers, inorganicnanoparticles, calcium phosphates, or a combination thereof.

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered bya combination of a vector and a non-vector based method. For example, avirosome comprises a liposome combined with an inactivated virus (e.g.,HIV or influenza virus), which can result in more efficient genetransfer, e.g., in a respiratory epithelial cell than either a viral ora liposomal method alone.

In an embodiment, the delivery vehicle is a non-viral vector. In anembodiment, the non-viral vector is an inorganic nanoparticle (e.g.,attached to the payload to the surface of the nanoparticle). Exemplaryinorganic nanoparticles include, e.g., magnetic nanoparticles (e.g., Fe1v1n0₂), or silica. The outer surface of the nanoparticle can beconjugated with a positively charged polymer (e.g., polyethylenimine,polylysine, polyserine) which allows for attachment (e.g., conjugationor entrapment) of payload. In an embodiment, the non-viral vector is anorganic nanoparticle (e.g., entrapment of the payload inside thenanoparticle). Exemplary organic nanoparticles include, e.g., SNALPliposomes that contain cationic lipids together with neutral helperlipids which are coated with polyethylene glycol (PEG) and protamine andnucleic acid complex coated with lipid coating.

Exemplary lipids and/or polymers for transfer of CRISPR systems ornucleic acid, e.g., vectors, encoding CRISPR systems or componentsthereof include, for example, those described in WO2011/076807,WO2014/136086, WO2005/060697, WO2014/140211, WO2012/031046,WO2013/103467, WO2013/006825, WO2012/006378, WO2015/095340, andWO2015/095346, the contents of each of the foregoing are herebyincorporated by reference in their entirety. In an embodiment, thevehicle has targeting modifications to increase target cell update ofnanoparticles and liposomes, e.g., cell specific antigens, monoclonalantibodies, single chain antibodies, aptamers, polymers, sugars, andcell penetrating peptides. In an embodiment, the vehicle uses fusogenicand endosome-destabilizing peptides/polymers. In an embodiment, thevehicle undergoes acid-triggered conformational changes (e.g., toaccelerate endosomal escape of the cargo). In an embodiment, astimuli-cleavable polymer is used, e.g., for release in a cellularcompartment. For example, disulfide-based cationic polymers that arecleaved in the reducing cellular environment can be used.

In an embodiment, the delivery vehicle is a biological non-viraldelivery vehicle. In an embodiment, the vehicle is an attenuatedbacterium (e.g., naturally or artificially engineered to be invasive butattenuated to prevent pathogenesis and expressing the transgene (e.g.,Listeria monocytogenes, certain Salmonella strains, Bifidobacteriumlongum, and modified Escherichia coli), bacteria having nutritional andtissue-specific tropism to target specific tissues, bacteria havingmodified surface proteins to alter target tissue specificity). In anembodiment, the vehicle is a genetically modified bacteriophage (e.g.,engineered phages having large packaging capacity, less immunogenic,containing mammalian plasmid maintenance sequences and havingincorporated targeting ligands) In an embodiment, the vehicle is amammalian virus-like particle. For example, modified viral particles canbe generated (e.g., by purification of the “empty” particles followed byex vivo assembly of the virus with the desired cargo). The vehicle canalso be engineered to incorporate targeting ligands to alter targettissue specificity. In an embodiment, the vehicle is a biologicalliposome. For example, the biological liposome is a phospholipid-basedparticle derived from human cells (e.g., erythrocyte ghosts, which arered blood cells broken down into spherical structures derived from thesubject (e.g., tissue targeting can be achieved by attachment of varioustissue or cell-specific ligands), or secretory exosomes—subject (i.e.,patient) derived membrane-bound nanovesicle (30-100 nm) of endocyticorigin (e.g., can be produced from various cell types and can thereforebe taken up by cells without the need of for targeting ligands).

In an embodiment, one or more nucleic acid molecules (e.g., DNAmolecules) other than the components of a Cas system, e.g., the Cas9molecule component and/or the gRNA molecule component described herein,are delivered. In an embodiment, the nucleic acid molecule is deliveredat the same time as one or more of the components of the Cas system aredelivered. In an embodiment, the nucleic acid molecule is deliveredbefore or after (e.g., less than about 30 minutes, 1 hour, 2 hours, 3hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2weeks, or 4 weeks) one or more of the components of the Cas9 system aredelivered. In an embodiment, the nucleic acid molecule is delivered by adifferent means than one or more of the components of the Cas9 system,e.g., the Cas9 molecule component and/or the gRNA molecule component,are delivered. The nucleic acid molecule can be delivered by any of thedelivery methods described herein. For example, the nucleic acidmolecule can be delivered by a viral vector, e.g., anintegration-deficient lentivirus, and the Cas9 molecule component and/orthe gRNA molecule component can be delivered by electroporation, e.g.,such that the toxicity caused by nucleic acids (e.g., DNAs) can bereduced. In an embodiment, the nucleic acid molecule encodes atherapeutic protein, e.g., a protein described herein. In an embodiment,the nucleic acid molecule encodes an RNA molecule, e.g., an RNA moleculedescribed herein. Delivery of RNA encoding a Cas9 molecule

RNA encoding Cas9 molecules (e.g., active Cas9 molecules, inactive Cas9molecules or inactive Cas9 fusion proteins) and/or gRNA molecules, canbe delivered into cells, e.g., target cells described herein, byart-known methods or as described herein. For example, Cas9-encodingand/or gRNA-encoding RNA can be delivered, e.g., by microinjection,electroporation, lipid-mediated transfection, peptide-mediated delivery,or a combination thereof.

Delivery of Cas9 Molecule as Protein

Cas9 molecules (e.g., active Cas9 molecules, inactive Cas9 molecules orinactive Cas9 fusion proteins) can be delivered into cells by art-knownmethods or as described herein. For example, Cas9 protein molecules canbe delivered, e.g., by microinjection, electroporation, lipid-mediatedtransfection, peptide-mediated delivery, cell squeezing or abrasion(e.g., by nanoneedles) or a combination thereof. Delivery can beaccompanied by DNA encoding a gRNA or by a gRNA, e.g., by precomplexingthe gRNA and the Cas9 protein in a ribonuclear protein complex (RNP).

In an aspect the Cas9 molecule, e.g., as described herein, is deliveredas a protein and the gRNA molecule is delivered as one or more RNAs(e.g., as a dgRNA or sgRNA, as described herein). In embodiments, theCas9 protein is complexed with the gRNA molecule prior to delivery to acell, e.g., as described herein, as a ribonuclear protein complex(“RNP”). In embodiments, the RNP can be delivered into cells, e.g.,described herein, by any art-known method, e.g., electroporation. Asdescribed herein, and without being bound by theory, it can bepreferable to use a gRNA molecule and Cas9 molecule which result in high% editing at the target sequence (e.g., >85%, >90%, >95%, >98%, or >99%)in the target cell, e.g., described herein, even when the concentrationof RNP delivered to the cell is reduced. Again, without being bound bytheory, delivering a reduced or low concentration of RNP comprising agRNA molecule that produces a high % editing at the target sequence inthe target cell (including at the low RNP concentration), can bebeneficial because it may reduce the frequency and number of off-targetediting events. In one aspect, where a low or reduced concentration ofRNP is to be used, the following exemplary procedure can be used togenerate the RNP with a dgRNA molecule:

-   -   1. Provide the Cas9 molecule and the tracr in solution at a high        concentration (e.g., a concentration higher than the final RNP        concentration to be delivered to the cell), and allow the two        components to equilibrate;    -   2. Provide the crRNA molecule, and allow the components to        equilibrate (thereby forming a high-concentration solution of        the RNP);    -   3. Dilute the RNP solution to the desired concentration;    -   4. Deliver said RNP at said desired concentration to the target        cells, e.g., by electroporation.

The above procedure may be modified for use with sgRNA molecules byomitting step 2, above, and in step 1, providing the Cas9 molecule andthe sgRNA molecule in solution at high concentration, and allowing thecomponents to equilibrate. In embodiments, the Cas9 molecule and eachgRNA component are provided in solution at a 1:2 ratio (Cas9:gRNA),e.g., a 1:2 molar ratio of Cas9:gRNA molecule. Where dgRNA molecules areused, the ratio, e.g., molar ratio, is 1:2:2 (Cas9:tracr:crRNA). Inembodiments, the RNP is formed at a concentration of 20 uM or higher,e.g., a concentration from about 20 uM to about 50 uM. In embodiments,the RNP is formed at a concentration of 10 uM or higher, e.g., aconcentration from about 10 uM to about 30 uM. In embodiments, the RNPis diluted to a final concentration of 10 uM or less (e.g., aconcentration from about 0.01 uM to about 10 uM) in a solutioncomprising the target cell (e.g., described herein) for delivery to saidtarget cell. In embodiments, the RNP is diluted to a final concentrationof 3 uM or less (e.g., a concentration from about 0.01 uM to about 3 uM)in a solution comprising the target cell (e.g., described herein) fordelivery to said target cell. In embodiments, the RNP is diluted to afinal concentration of 1 uM or less (e.g., a concentration from about0.01 uM to about 1 uM) in a solution comprising the target cell (e.g.,described herein) for delivery to said target cell. In embodiments, theRNP is diluted to a final concentration of 0.3 uM or less (e.g., aconcentration from about 0.01 uM to about 0.3 uM) in a solutioncomprising the target cell (e.g., described herein) for delivery to saidtarget cell. In embodiments, the RNP is provided at a finalconcentration of about 3 uM in a solution comprising the target cell(e.g., described herein) for delivery to said target cell. Inembodiments, the RNP is provided at a final concentration of about 2 uMin a solution comprising the target cell (e.g., described herein) fordelivery to said target cell. In embodiments, the RNP is provided at afinal concentration of about 1 uM in a solution comprising the targetcell (e.g., described herein) for delivery to said target cell. Inembodiments, the RNP is provided at a final concentration of about 0.3uM in a solution comprising the target cell (e.g., described herein) fordelivery to said target cell. In embodiments, the RNP is provided at afinal concentration of about 0.1 uM in a solution comprising the targetcell (e.g., described herein) for delivery to said target cell. Inembodiments, the RNP is provided at a final concentration of about 0.05uM in a solution comprising the target cell (e.g., described herein) fordelivery to said target cell. In embodiments, the RNP is provided at afinal concentration of about 0.03 uM in a solution comprising the targetcell (e.g., described herein) for delivery to said target cell. Inembodiments, the RNP is provided at a final concentration of about 0.01uM in a solution comprising the target cell (e.g., described herein) fordelivery to said target cell. In embodiments, the RNP is formulated in amedium suitable for electroporation. In embodiments, the RNP isdelivered to cells, e.g., HSPC cells, e.g., as described herein, byelectroporation, e.g., using electroporation conditions describedherein.

In aspects, the components of the gene editing system (e.g., CRISPRsystem) and/or nucleic acid encoding one or more components of the geneediting system (e.g., CRISPR system) are introduced into the cells bymechanically perturbing the cells, for example, by passing said cellsthrough a pore or channel which constricts the cells. Such perturbationmay be accomplished in a solution comprising the components of the geneediting system (e.g., CRISPR system) and/or nucleic acid encoding one ormore components of the gene editing system (e.g., CRISPR system), e.g.,as described herein. In embodiments, the perturbation is accomplishedusing a TRIAMF system, e.g., as described herein, for example, in theExamples and in PCT patent application PCT/US17/54110 (incorporatedherein by reference in its entirety).

Bi-Modal or Differential Delivery of Components

Separate delivery of the components of a Cas system, e.g., the Cas9molecule component and the gRNA molecule component, and moreparticularly, delivery of the components by differing modes, can enhanceperformance, e.g., by improving tissue specificity and safety.

In an embodiment, the Cas9 molecule and the gRNA molecule are deliveredby different modes, or as sometimes referred to herein as differentialmodes. Different or differential modes, as used herein, refer modes ofdelivery that confer different pharmacodynamic or pharmacokineticproperties on the subject component molecule, e.g., a Cas9 molecule,gRNA molecule, or template nucleic acid. For example, the modes ofdelivery can result in different tissue distribution, differenthalf-life, or different temporal distribution, e.g., in a selectedcompartment, tissue, or organ.

Some modes of delivery, e.g., delivery by a nucleic acid vector thatpersists in a cell, or in progeny of a cell, e.g., by autonomousreplication or insertion into cellular nucleic acid, result-in morepersistent expression of and presence of a component.

XI. Methods of Treatment

The Cas9 systems, e.g., one or more gRNA molecules and one or more Cas9molecules, described herein are useful for the treatment of disease in amammal, e.g., in a human. The terms “treat,” “treated,” “treating,” and“treatment,” include the administration of cas9 systems, e.g., one ormore gRNA molecules and one or more cas9 molecules, to cells to preventor delay the onset of the symptoms, complications, or biochemicalindicia of a disease, alleviating the symptoms or arresting orinhibiting further development of the disease, condition, or disorder.Treatment may also include the administration of one or more (e.g., apopulation of) cells, e.g., HSPCs, that have been modified by theintroduction of a gRNA molecule (or more than one gRNA molecule) of thepresent invention, or by the introduction of a CRISPR system asdescribed herein, or by any of the methods of preparing said cellsdescribed herein, to prevent or delay the onset of the symptoms,complications, or biochemical indicia of a disease, alleviating thesymptoms or arresting or inhibiting further development of the disease,condition, or disorder. Treatment may be prophylactic (to prevent ordelay the onset of the disease, or to prevent the manifestation ofclinical or subclinical symptoms thereof) or therapeutic suppression oralleviation of symptoms after the manifestation of the disease.Treatment can be measured by the therapeutic measures described herein.Thus, the methods of “treatment” of the present invention also includeadministration of cells altered by the introduction of a cas9 system(e.g., one or more gRNA molecules and one or more Cas9 molecules) intosaid cells to a subject in order to cure, reduce the severity of, orameliorate one or more symptoms of a disease or condition, in order toprolong the health or survival of a subject beyond that expected in theabsence of such treatment. For example, “treatment” includes thealleviation of a disease symptom in a subject by at least 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.

Cas9 systems comprising gRNA molecules comprising the targeting domainsdescribed herein, e.g., in Table 1, and the methods and cells (e.g., asdescribed herein) are useful for the treatment of hemoglobinopathies.

Hemoglobinopathies

Hemoglobinopathies encompass a number of anemias of genetic origin inwhich there is a decreased production and/or increased destruction(hemolysis) of red blood cells (RBCs). These also include geneticdefects that result in the production of abnormal hemoglobins with aconcomitant impaired ability to maintain oxygen concentration. Some suchdisorders involve the failure to produce normal β-globin in sufficientamounts, while others involve the failure to produce normal β-globinentirely. These disorders associated with the β-globin protein arereferred to generally as β-hemoglobinopathies. For example,β-thalassemias result from a partial or complete defect in theexpression of the β-globin gene, leading to deficient or absent HbA.Sickle cell anemia results from a point mutation in the β-globinstructural gene, leading to the production of an abnormal (sickle)hemoglobin (HbS). HbS is prone to polymerization, particularly underdeoxygenated conditions. HbS RBCs are more fragile than normal RBCs andundergo hemolysis more readily, leading eventually to anemia.

In an embodiment, a genetic defect in alpha globin or beta globin iscorrected, e.g., by homologous recombination, using the Cas9 moleculesand gRNA molecules, e.g., CRISPR systems, described herein.

In an embodiment, a gene encoding a wild type (e.g., non-mutated) copyof alpha globin or beta globin is inserted into the genome of the cell,e.g., at a safe harbor site, e.g., at an AAVS1 safe harbor site, byhomologous recombination, using a CRISPR system and methods describedherein.

In an embodiment, a hemoglobinopathies-associated gene is targeted,using the Cas9 molecule and gRNA molecule described herein. Exemplarytargets include, e.g., genes associated with control of the gamma-globingenes. In an embodiment, the target is a nondeletional HPFH region.

Fetal hemoglobin (also hemoglobin F or HbF or α2γ2) is a tetramer of twoadult alpha-globin polypeptides and two fetal beta-like gamma-globinpolypeptides. HbF is the main oxygen transport protein in the humanfetus during the last seven months of development in the uterus and inthe newborn until roughly 6 months old. Functionally, fetal hemoglobindiffers most from adult hemoglobin in that it is able to bind oxygenwith greater affinity than the adult form, giving the developing fetusbetter access to oxygen from the mother's bloodstream.

In newborns, fetal hemoglobin is nearly completely replaced by adulthemoglobin by approximately 6 months postnatally. In adults, fetalhemoglobin production can be reactivated pharmacologically, which isuseful in the treatment of diseases such as hemoglobinopathies. Forexample, in certain patients with hemoglobinopathies, higher levels ofgamma-globin expression can partially compensate for defective orimpaired beta-globin gene production, which can ameliorate the clinicalseverity in these diseases. Increased HbF levels or F-cell (HbFcontaining erythrocyte) numbers can ameliorate the disease severity ofhemoglobinopathies, e.g., beta-thalassemia major and sickle cell anemia.

As was surprisingly discovered, increased HbF levels or F-cell countscan be associated indel formation at one or more nondeltional HPFHregions in cells, for example, HSPCs and/or cells differentiated fromHSPCs (e.g., HSPCs modified by one or more gRNA molecules describedherein). In an embodiment, the cell is a hemopoietic stem cell orprogenitor cell.

Sickle Cell Diseases

Sickle cell disease is a group of disorders that affects hemoglobin.People with this disorder have atypical hemoglobin molecules (hemoglobinS), which can distort red blood cells into a sickle, or crescent, shape.Characteristic features of this disorder include a low number of redblood cells (anemia), repeated infections, and periodic episodes ofpain.

Mutations in the HBB gene cause sickle cell disease. The HBB geneprovides instructions for making beta-globin. Various versions ofbeta-globin result from different mutations in the HBB gene. Oneparticular HBB gene mutation produces an abnormal version of beta-globinknown as hemoglobin S (HbS). Other mutations in the HBB gene lead toadditional abnormal versions of beta-globin such as hemoglobin C (HbC)and hemoglobin E (HbE). HBB gene mutations can also result in anunusually low level of beta-globin, i.e., beta thalassemia.

In people with sickle cell disease, at least one of the beta-globinsubunits in hemoglobin is replaced with hemoglobin S. In sickle cellanemia, which is a common form of sickle cell disease, hemoglobin Sreplaces both beta-globin subunits in hemoglobin. In other types ofsickle cell disease, just one beta-globin subunit in hemoglobin isreplaced with hemoglobin S. The other beta-globin subunit is replacedwith a different abnormal variant, such as hemoglobin C. For example,people with sickle-hemoglobin C (HbSC) disease have hemoglobin moleculeswith hemoglobin S and hemoglobin C instead of beta-globin. If mutationsthat produce hemoglobin S and beta thalassemia occur together,individuals have hemoglobin S-beta thalassemia (HbSBetaThal) disease.

Beta Thalassemia

Beta thalassemia is a blood disorder that reduces the production ofhemoglobin. In people with beta thalassemia, low levels of hemoglobinlead to a lack of oxygen in many parts of the body. Affected individualsalso have a shortage of red blood cells (anemia), which can cause paleskin, weakness, fatigue, and more serious complications. People withbeta thalassemia are at an increased risk of developing abnormal bloodclots.

Beta thalassemia is classified into two types depending on the severityof symptoms: thalassemia major (also known as Cooley's anemia) andthalassemia intermedia. Of the two types, thalassemia major is moresevere.

Mutations in the HBB gene cause beta thalassemia. The HBB gene providesinstructions for making beta-globin. Some mutations in the HBB geneprevent the production of any beta-globin. The absence of beta-globin isreferred to as beta-zero (B^(o)) thalassemia. Other HBB gene mutationsallow some beta-globin to be produced but in reduced amounts, i.e.,beta-plus (B⁺) thalassemia. People with both types have been diagnosedwith thalassemia major and thalassemia intermedia.

In an embodiment, a Cas9 molecule/gRNA molecule complex targeting afirst gene or locus is used to treat a disorder characterized by asecond gene, e.g., a mutation in a second gene. By way of example,targeting of the first gene, e.g., by editing or payload delivery, cancompensate for, or inhibit further damage from, the affect of a secondgene, e.g., a mutant second gene. In an embodiment the allele(s) of thefirst gene carried by the subject is not causative of the disorder. Forexample, as shown herein, gRNA molecules which induce indel formation ata nondeletional HPFH region, for example an HBG1 and/or HBG2 promoterregion, can result in upregulation of fetal hemoglobin in erythroidcells differentiated from modified HSPCs (as described herein), andwithout being bound by theory, such fetal hemoglobin upregulationcompensates and corrects for the HBB gene harboring a sickle mutation.

In one aspect, the invention relates to the treatment of a mammal, e.g.,a human, in need of increased fetal hemoglobin (HbF).

In one aspect, the invention relates to the treatment of a mammal, e.g.,a human, that has been diagnosed with, or is at risk of developing, ahemoglobinopathy.

In one aspect, the hemoglobinopathy is a β-hemoglobinopathy. In oneaspect, the hemoglobinopathy is sickle cell disease. In one aspect, thehemoglobinopathy is beta thalassemia.

Methods of Treatment of Hemoglobinopathies

In another aspect the invention provides methods of treatment. Inaspects, the gRNA molecules, CRISPR systems and/or cells of theinvention are used to treat a patient in need thereof. In aspects, thepatient is a mammal, e.g., a human. In aspects, the patient has ahemoglobinopathy. In embodiments, the patient has sickle cell disease.In embodiments, the patient has beta thalassemia.

In one aspect, the method of treatment comprises administering to amammal, e.g., a human, one or more gRNA molecules, e.g., one or moregRNA molecules comprising a targeting domain described in Table 1, andone or more cas9 molecules described herein.

In one aspect, the method of treatment comprises administering to amammal a cell population, wherein the cell population is a cellpopulation from a mammal, e.g., a human, that has been administered oneor more gRNA molecules, e.g., one or more gRNA molecules comprising atargeting domain described in Table 1, and one or more cas9 moleculesdescribed herein, e.g., a CRISPR system as described herein. In oneembodiment, the administration of the one or more gRNA molecules orCRISPR systems to the cell is accomplished in vivo. In one embodimentthe administration of the one or more gRNA molecules or CRISPR systemsto the cell is accomplished ex vivo.

In one aspect, the method of treatment comprises administering to themammal, e.g., the human, an effective amount of a cell populationcomprising cells which comprise or at one time comprised one or moregRNA molecules, e.g., one or more gRNA molecules comprising a targetingdomain described in Table 1, and one or more cas9 molecules describedherein, or the progeny of said cells. In one embodiment, the cells areallogeneic to the mammal. In one embodiment, the cells are autologous tothe mammal. In one embodiment the cells are harvested from the mammal,manipulated ex vivo, and returned to the mammal.

In aspects, the cells comprising or which at one time comprised one ormore gRNA molecules, e.g., one or more gRNA molecules comprising atargeting domain described in Table 1, and one or more cas9 moleculesdescribed herein, or the progeny of said cells, comprise stem cells orprogenitor cells. In one aspect, the stem cells are hematopoietic stemcells. In one aspect, the progenitor cells are hematopoietic progenitorcells. In one aspect, the cells comprise both hematopoietic stem cellsand hematopoietic progenitor cells, e.g., are HSPCs. In one aspect, thecells comprise, e.g., consist of, CD34+ cells. In one aspect the cellsare substantially free of CD34− cells. In one aspect, the cellscomprise, e.g., consist of, CD34+/CD90+ stem cells. In one aspect, thecells comprise, e.g., consist of, CD34+/CD90− cells. In an aspect, thecells are a population comprising one or more of the cell typesdescribed above or described herein.

In one embodiment, the disclosure provides a method for treating ahemoglobinopathy, e.g., sickle cell disease or beta-thalassemia, or amethod for increasing fetal hemoglobin expression in a mammal, e.g., ahuman, in need thereof, the method comprising:

a) providing, e.g., harvesting or isolating, a population of HSPCs(e.g., CD34+ cells) from a mammal;

b) providing said cells ex vivo, e.g., in a cell culture medium,optionally in the presence of an effective amount of a compositioncomprising at least one stem cell expander, whereby said population ofHSPCs (e.g., CD34+ cells) expands to a greater degree than an untreatedpopulation;

c) contacting the population of HSPCs (e.g., CD34+ cells) with aneffective amount of: a composition comprising at least one gRNA moleculecomprising a targeting domain described herein, e.g., a targeting domaindescribed in Table 1, or a nucleic acid encoding said gRNA molecule, andat least one cas9 molecule, e.g., described herein, or a nucleic acidencoding said cas9 molecule, e.g., one or more RNPs as described herein,e.g., with a CRISPR system described herein;

d) causing at least one modification in at least a portion of the cellsof the population (e.g., at least a portion of the HSPCs, e.g., CD34+cells, of the population), whereby, e.g., when said HSPCs aredifferentiated into cells of an erythroid lineage, e.g., red bloodcells, fetal hemoglobin expression is increased, e.g., relative to cellsnot contacted according to step c); and

f) returning a population of cells comprising said modified HSPCs (e.g.,CD34+ cells) to the mammal.

In an aspect, the HSPCs are allogeneic to the mammal to which they arereturned. In an aspect, the HSPCs are autologous to the mammal to whichthey are returned. In aspects, the HSPCs are isolated from bone marrow.In aspects, the HSPCs are isolated from peripheral blood, e.g.,mobilized peripheral blood. In aspects, the mobilized peripheral bloodis isolated from a subject who has been administered a G-CSF. Inaspects, the mobilized peripheral blood is isolated from a subject whohas been administered a mobilization agent other than G-CSF, forexample, Plerixafor® (AMD3100). In other aspects, the mobilizedperipheral blood is isolated from a subject who has been administered acombination of G-CSF and Plerixafor® (AMD3100)). In aspects, the HSPCsare isolated from umbilical cord blood. In embodiments, the cells arederived from a hemoglobinopathy patient, for example a patient withsickle cell disease or a patient with a thalassemia, e.g.,beta-thalassemia.

In further embodiments of the method, the method further comprises,after providing a population of HSPCs (e.g., CD34+ cells), e.g., from asource described above, the step of enriching the population of cellsfor HSPCs (e.g., CD34+ cells). In embodiments of the method, after saidenriching, the population of cells, e.g., HSPCs, is substantially freeof CD34− cells.

In embodiments, the population of cells which is returned to the mammalincludes at least 70% viable cells. In embodiments, the population ofcells which is returned to the mammal includes at least 75% viablecells. In embodiments, the population of cells which is returned to themammal includes at least 80% viable cells. In embodiments, thepopulation of cells which is returned to the mammal includes at least85% viable cells. In embodiments, the population of cells which isreturned to the mammal includes at least 90% viable cells. Inembodiments, the population of cells which is returned to the mammalincludes at least 95% viable cells. In embodiments, the population ofcells which is returned to the mammal includes at least 99% viablecells. Viability can be determined by staining a representative portionof the population of cells for a cell viability marker, e.g., as knownin the art.

In another embodiment, the disclosure provides a method for treating ahemoglobinopathy, e.g., sickle cell disease or beta-thalassemia, or amethod for increasing fetal hemoglobin expression in a mammal, e.g., ahuman, in need thereof, the method comprising the steps of:

a) providing, e.g., harvesting or isolating, a population of HSPCs(e.g., CD34+ cells) of a mammal, e.g., from the bone marrow of a mammal;

b) isolating the CD34+ cells from the population of cells of step a);

c) providing said CD34+ cells ex vivo, and culturing said cells, e.g.,in a cell culture medium, in the presence of an effective amount of acomposition comprising at least one stem cell expander, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-olat a concentration of about 0.5 to about 0.75 micromolar, whereby saidpopulation of CD34+ cells expands to a greater degree than an untreatedpopulation;

d) introducing into the cells of the population CD34+ cells an effectiveamount of: a composition comprising a Cas9 molecule, e.g., as describedherein, and a gRNA molecule, e.g., as described herein, e.g., optionallywhere the Cas9 molecule and the gRNA molecule are in the form of an RNP,e.g., as described herein, and optionally where said introduction is byelectroporation, e.g., as described herein, of said RNP into said cells;

e) causing at least one genetic modification in at least a portion ofthe cells of the population (e.g., at least a portion of the HSPCs,e.g., CD34+ cells, of the population), whereby an indel, e.g., asdescribed herein, is created at or near the genomic site complementaryto the targeting domain of the gRNA introduced in step d);

f) optionally, additionally culturing said cells after said introducing,e.g., in a cell culture medium, in the presence of an effective amountof a composition comprising at least one stem cell expander, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-olat a concentration of about 0.5 to about 0.75 micromolar, such that thecells expand at least 2-fold, e.g., at least 4-fold, e.g., at least5-fold;

g) cryopreserving said cells; and

h) returning the cells to the mammal, wherein,

-   -   the cells returned to the mammal comprise cells that 1) maintain        the ability to differentiate into cells of the erythroid        lineage, e.g., red blood cells; 2) when differentiated into red        blood cells, produce an increased level of fetal hemoglobin,        e.g., relative to cells unmodified by the gRNA of step e), e.g.,        produce at least 6 picograms fetal hemoglobin per cell.

In an aspect, the HSPCs are allogeneic to the mammal to which they arereturned. In an aspect, the HSPCs are autologous to the mammal to whichthey are returned. In aspects, the HSPCs are isolated from bone marrow.In aspects, the HSPCs are isolated from peripheral blood, e.g.,mobilized peripheral blood. In aspects, the mobilized peripheral bloodis isolated from a subject who has been administered a G-CSF. Inaspects, the mobilized peripheral blood is isolated from a subject whohas been administered a mobilization agent other than G-CSF, forexample, Plerixafor® (AMD3100). In other aspects, the mobilizedperipheral blood is isolated from a subject who has been administered acombination of G-CSF and Plerixafor® (AMD3100)). In aspects, the HSPCsare isolated from umbilical cord blood. In embodiments, the cells arederived from a hemoglobinopathy patient, for example a patient withsickle cell disease or a patient with a thalassemia, e.g.,beta-thalassemia.

In embodiments of the method above, the recited step b) results in apopulation of cells which is substantially free of CD34− cells.

In further embodiments of the method, the method further comprises,after providing a population of HSPCs (e.g., CD34+ cells), e.g., from asource described above, the population of cells is enriched for HSPCs(e.g., CD34+ cells).

In a further embodiments of these methods, the population of modifiedHSPCs (e.g., CD34+ stem cells) having the ability to differentiateincreased fetal hemoglobin expression is cryopreserved and stored priorto being reintroduced into the mammal. In embodiments, the cryopreservedpopulation of HSPCs having the ability to differentiate into cells ofthe erythroid lineage, e.g., red blood cells, and/or when differentiatedinto cells of the erythroid lineage, e.g., red blood cells, produce anincreased level of fetal hemoglobin is thawed and then reintroduced intothe mammal. In a further embodiment of these methods, the methodcomprises chemotherapy and/or radiation therapy to remove or reduce theendogenous hematopoietic progenitor or stem cells in the mammal. In afurther embodiment of these methods, the method does not comprise a stepof chemotherapy and/or radiation therapy to remove or reduce theendogenous hematopoietic progenitor or stem cells in the mammal. In afurther embodiment of these methods, the method comprises a chemotherapyand/or radiation therapy to reduce partially (e.g., partiallymphodepletion) the endogenous hematopoietic progenitor or stem cellsin the mammal. In embodiments the patient is treated with a fullylymphodepleting dose of busulfan prior to reintroduction of the modifiedHSPCs to the mammal. In embodiments, the patient is treated with apartially lymphodepleting dose of busulfan prior to reintroduction ofthe modified HSPCs to the mammal.

In embodiments, the cells are contacted with RNP comprising a Cas9molecule, e.g., as described herein, complexed with a gRNA to anondeletional HPFH region, e.g., as described herein (e.g., comprising atargeting domain listed in Table 1.

In embodiments, the stem cell expander is Compound 1. In embodiments,the stem cell expander is Compound 2. In embodiments, the stem cellexpander is Compound 3. In embodiments, the stem cell expander is(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol.In embodiments, the stem cell expander is(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-oland is present at a concentration of 2-0.1 micromolar, e.g., 1-0.25micromolar, e.g., 0.75-0.5 micromolar. In embodiments, the stem cellexpander is a molecule described in WO2010/059401 (e.g., the moleculedescribed in Example 1 of WO2010/059401).

In embodiments, the cells, e.g., HSPCs, e.g., as described herein, arecultured ex vivo for a period of about 1 hour to about 15 days, e.g., aperiod of about 12 hours to about 12 days, e.g., a period of about 12hours to 4 days, e.g., a period of about 1 day to about 4 days, e.g., aperiod of about 1 day to about 2 days, e.g., a period of about 1 day ora period of about 2 days, prior to the step of contacting the cells witha CRISPR system, e.g., described herein. In embodiments, said culturingprior to said contacting step is in a composition (e.g., a cell culturemedium) comprising a stem cell expander, e.g., described herein, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-olat a concentration of about 0.25 uM to about 1 uM, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-olat a concentration of about 0.75-0.5 micromolar. In embodiments, thecells are cultured ex vivo for a period of no more than about about 1day, e.g., no more than about 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,7, 6, 5, 4, 3, 2, or 1 hour(s) after the step of contacting the cellswith a CRISPR system, e.g., described herein, e.g., in a cell culturemedium which comprises a stem cell expander, e.g., described herein,e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-olat a concentration of about 0.25 uM to about 1 uM, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-olat a concentration of about 0.75-0.5 micromolar. In other embodiments,the cells are cultured ex vivo for a period of about 1 hour to about 15days, e.g., a period of about 12 hours to about 10 days, e.g., a periodof about 1 day to about 10 days, e.g., a period of about 1 day to about5 days, e.g., a period of about 1 day to about 4 days, e.g., a period ofabout 2 days to about 4 days, e.g., a period of about 2 days, about 3days or about 4 days, after the step of contacting the cells with aCRISPR system, e.g., described herein, in a cell culture medium, e.g.,which comprises a stem cell expander, e.g., described herein, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-olat a concentration of about 0.25 uM to about 1 uM, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-olat a concentration of about 0.75-0.5 micromolar. In embodiments, thecells are cultured ex vivo (e.g., cultured prior to said contacting stepand/or cultured after said contacting step) for a period of about 1 hourto about 20 days, e.g., a period of about 6-12 days, e.g., a period ofabout 6, about 7, about 8, about 9, about 10, about 11, or about 12days.

In embodiments, the population of cells comprising the modified HSPCsreturned to the mammal comprises at least about 1 million cells (e.g.,at least about 1 million CD34+ cells) per kg. In embodiments, thepopulation of cells comprising the modified HSPCs returned to the mammalcomprises at least about 2 million cells (e.g., at least about 2 millionCD34+ cells) per kg. In embodiments, the population of cells comprisingthe modified HSPCs returned to the mammal comprises at least about 3million cells (e.g., at least about 3 million CD34+ cells) per kg. Inembodiments, the population of cells comprising the modified HSPCsreturned to the mammal comprises at least about 4 million cells (e.g.,at least about 4 million CD34+ cells) per kg. In embodiments, thepopulation of cells comprising the modified HSPCs returned to the mammalcomprises at least about 5 million cells (e.g., at least about 5 millionCD34+ cells) per kg. In embodiments, the population of cells comprisingthe modified HSPCs returned to the mammal comprises at least about 6million cells (e.g., at least about 6 million CD34+ cells) per kg. Inembodiments, the population of cells comprising the modified HSPCsreturned to the mammal comprises at least 1 million cells (e.g., atleast 1 million CD34+ cells) per kg. In embodiments, the population ofcells comprising the modified HSPCs returned to the mammal comprises atleast 2 million cells (e.g., at least 2 million CD34+ cells) per kg. Inembodiments, the population of cells comprising the modified HSPCsreturned to the mammal comprises at least 3 million cells (e.g., atleast 3 million CD34+ cells) per kg. In embodiments, the population ofcells comprising the modified HSPCs returned to the mammal comprises atleast 4 million cells (e.g., at least 4 million CD34+ cells) per kg. Inembodiments, the population of cells comprising the modified HSPCsreturned to the mammal comprises at least 5 million cells (e.g., atleast 5 million CD34+ cells) per kg. In embodiments, the population ofcells comprising the modified HSPCs returned to the mammal comprises atleast 6 million cells (e.g., at least 6 million CD34+ cells) per kg. Inembodiments, the population of cells comprising the modified HSPCsreturned to the mammal comprises about 1 million cells (e.g., about 1million CD34+ cells) per kg. In embodiments, the population of cellscomprising the modified HSPCs returned to the mammal comprises about 2million cells (e.g., about 2 million CD34+ cells) per kg. Inembodiments, the population of cells comprising the modified HSPCsreturned to the mammal comprises about 3 million cells (e.g., about 3million CD34+ cells) per kg. In embodiments, the population of cellscomprising the modified HSPCs returned to the mammal comprises about 4million cells (e.g., about 4 million CD34+ cells) per kg. Inembodiments, the population of cells comprising the modified HSPCsreturned to the mammal comprises about 5 million cells (e.g., about 5million CD34+ cells) per kg. In embodiments, the population of cellscomprising the modified HSPCs returned to the mammal comprises about 6million cells (e.g., about 6 million CD34+ cells) per kg. Inembodiments, the population of cells comprising the modified HSPCsreturned to the mammal comprises about 2×10⁶ cells (e.g., about 2×10⁶CD34+ cells) per kg body weight of the patient. In embodiments, thepopulation of cells comprising the modified HSPCs returned to the mammalcomprises at least 2×10⁶ cells (e.g., about 2×10⁶ CD34+ cells) per kgbody weight of the patient. In embodiments, the population of cellscomprising the modified HSPCs returned to the mammal comprises between2×10⁶ cells (e.g., about 2×10⁶ CD34+ cells) per kg body weight of thepatient and 10×10⁶ cells (e.g., about 2×10⁶ CD34+ cells) per kg bodyweight of the patient. In embodiments, the cells comprising the modifiedcells are infused into the patient. In embodiments, before the cellscomprising the modified HSPCs are infused into the patient, the patientis treated with a lymphodepleting therapy, for example, is treated withbusulphan, for example is treated with a full lymphodepleting busulphanregimen, or for example is treated with a reduced intensity busulphanlymphodepleting regimen.

In embodiments, any of the methods described above results in thepatient having at least 80% of its circulating CD34+ cells comprising anindel at or near the genomic site complementary to the targeting domainof the gRNA molecule used in the method, e.g., as measured at least 15days, e.g., at least 20, at least 30, at least 40 at least 50 or atleast 60 days after reintroduction of the cells into the mammal Withoutbeing bound by theory, it has surprisingly been discovered herein thatindels and indel patterns (including large deletions) observed when geneediting systems, e.g., CRISPR systems, e.g., CRISPR systems comprising agRNA molecule targeting the HBG1 and/or HBG2 region, e.g., as describedherein, are introduced into HSPCs, and those cells are transplanted intoorganisms, certain gRNAs produce cells comprising indels and indelpatterns (including large indels) that remain detectible in the editedcell population and its progeny, in the organism, and persist for morethan 8 weeks, 12 weeks, 16 weeks or 20 weeks. Without being bound bytheory, a cell population comprising an indel pattern or particularindel (including large deletion) that persists within a detectible cellpopulation, for example, longer than 16 weeks or longer than 20 weeksafter introduction into an organism (e.g., a patient), could bebeneficial to producing a longer-term amelioration of a disease orcondition, e.g. described herein (e.g., a hemoglobinopathy, e.g., sicklecell disease or a thalassemia) than cells (or their progeny) that uponintroduction into an organism or patient lose one or more indels(including large deletions). In embodiments, the persisting indel orindel pattern is associated with upregulated fetal hemoglobin (e.g., inerythroid progeny of said cells). Thus, in embodiments, the presentdisclosure provides populations of cells, e.g., HSPCs, e.g., asdescribed herein, which comprise one or more indels (including largedeletions) which persist (e.g., remain detectible, e.g., in a cellpopulation or its progeny) in the blood and/or bone marrow) for morethan 8 weeks, more than 12 weeks, more than 16 weeks or more than 20weeks after introduction into an organism, e.g., patient.

In embodiments, any of the methods described above results in thepatient having at least 20% of its bone marrow CD34+ cells comprising anindel at or near the genomic site complementary to the targeting domainof the gRNA molecule used in the method, e.g., as measured at least 15days, e.g., at least 20, at least 30, at least 40 at least 50 or atleast 60 days after reintroduction of the cells into the mammal.

In embodiments, the HSPCs that are reintroduced into the mammal are ableto differentiate in vivo into cells of the erythroid lineage, e.g., redblood cells, and said differentiated cells exhibit increased fetalhemoglobin levels, e.g., produce at least 6 picograms fetal hemoglobinper cell, e.g., at least 7 picograms fetal hemoglobin per cell, at least8 picograms fetal hemoglobin per cell, at least 9 picograms fetalhemoglobin per cell, at least 10 picograms fetal hemoglobin per cell,e.g., between about 9 and about 10 picograms fetal hemoglobin per cell,e.g., such that the hemoglobinopathy is treated the mammal.

It will be understood that when a cell is characterized as havingincreased fetal hemoglobin, that includes embodiments in which aprogeny, e.g., a differentiated progeny, of that cell exhibits increasedfetal hemoglobin. For example, in the methods described herein, thealtered or modified CD34+ cell (or cell population) may not expressincreased fetal hemoglobin, but when differentiated into cells oferythroid lineage, e.g., red blood cells, the cells express increasedfetal hemoglobin, e.g., increased fetal hemoglobin relative to anunmodified or unaltered cell under similar conditions.

XII. Culture Methods and Methods of Manufacturing Cells

The disclosure provides methods of culturing cells, e.g., HSPCs, e.g.,hematopoietic stem cells, e.g., CD34+ cells modified, or to be modified,with the gRNA molecules described herein.

DNA Repair Pathway Inhibitors

Without being bound by theory, it is believed that the pattern of indelsproduced by a given gRNA molecule at a particular target sequence is aproduct of each of the active DNA repair mechanisms within the cell(e.g., non-homologous end joining, microhomology-mediated end joining,etc.). Without being bound by theory, it is believed that a particularlyfavorable indel may be selected for or enriched for by contacting thecells to be edited with an inhibitor of a DNA repair pathway that doesnot produce the desired indel. Thus, the gRNA molecules, CRISPR systems,methods and other aspects of the invention may be performed incombination with such inhibitors. Examples of such inhibitors includethose described in, e.g., WO2014/130955, the contents of which arehereby incorporated by reference in their entirety. In embodiment, theinhibitor is a DNAPKc inhibitor, e.g., NU7441.

Stem Cell Expanders

In one aspect the invention relates to culturing the cells, e.g., HSPCs,e.g., CD34+ cells modified, or to be modified, with the gRNA moleculesdescribed herein, with one or more agents that result in an increasedexpansion rate, increased expansion level, or increased engraftmentrelative to cells not treated with the agent. Such agents are referredto herein as stem cell expanders.

In an aspect, the one or more agents that result in an increasedexpansion rate or increased expansion level, relative to cells nottreated with the agent, e.g., the stem cell expander, comprises an agentthat is an inhibitor of the aryl hydrocarbon receptor (AHR) pathway. Inaspects, the stem cell expander is a compound disclosed in WO2013/110198or a compound disclosed in WO2010/059401, the contents of which areincorporated by reference in their entirety.

In one aspect, the one or more agents that result in an increasedexpansion rate or increased expansion level, relative to cells nottreated with the agent, is a pyrimido[4,5-b]indole derivative, e.g., asdisclosed in WO2013/110198, the contents of which are herebyincorporated by reference in their entirety. In one embodiment the agentis compound 1((1r,4r)—N¹-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamine):

In another aspect, the agent is Compound 2 (methyl4-(3-piperidin-1-ylpropylamino)-9H-pyrimido[4,5-b]indole-7-carboxylate):

In another aspect, the one or more agents that result in an increasedexpansion rate or increased expansion level, relative to cells nottreated with the agent, is an agent disclosed in WO2010/059401, thecontents of which are hereby incorporated by reference in theirentirety.

In one embodiment, the stem cell expander is compound 3:4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol,i.e., is the compound from example 1 of WO2010/059401, having thefollowing structure:

In another aspect, the stem cell expander is(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol((S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,i.e., is the compound 157S according to WO2010/059401), having thefollowing structure:

(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol

In embodiments the population of HSPCs is contacted with the stem cellexpander, e.g., compound 1, compound 2, compound 3,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,or combinations thereof (e.g., a combination of compound 1 and(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol)before introduction of the CRISPR system (e.g., gRNA molecule and/orCas9 molecule of the invention) to said HSPCs. In embodiments, thepopulation of HSPCs is contacted with the stem cell expander, e.g.,compound 1, compound 2, compound 3,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,or combinations thereof (e.g., a combination of compound 1 and(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol),after introduction of the CRISPR system (e.g., gRNA molecule and/or Cas9molecule of the invention) to said HSPCs. In embodiments, the populationof HSPCs is contacted with the stem cell expander, e.g., compound 1,compound 2, compound 3,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,or combinations thereof (e.g., a combination of compound 1 and(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol),both before and after introduction of the CRISPR system (e.g., gRNAmolecule and/or Cas9 molecule of the invention) to said HSPCs.

In embodiments, the stem cell expander is present in an effective amountto increase the expansion level of the HSPCs, relative to HSPCs in thesame media but for the absence of the stem cell expander. Inembodiments, the stem cell expander is present at a concentrationranging from about 0.01 to about 10 uM, e.g., from about 0.1 uM to about1 uM. In embodiments, the stem cell expander is present in the cellculture medium at a concentration of about 1 uM, about 950 nM, about 900nM, about 850 nM, about 800 nM, about 750 nM, about 700 nM, about 650nM, about 600 nM, about 550 nM, about 500 nM, about 450 nM, about 400nM, about 350 nM, about 300 nM, about 250 nM, about 200 nM, about 150nM, about 100 nM, about 50 nM, about 25 nM, or about 10 nM. Inembodiments, the stem cell expander is present at a concentrationranging from about 500 nM to about 750 nM.

In embodiments, the stem cell expander is(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,which is present in the cell culture medium at a concentration rangingfrom about 0.01 to about 10 micromolar (uM). In embodiments, the stemcell expander is(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,which is present in the cell culture medium at a concentration rangingfrom about 0.1 to about 1 micromolar (uM). In embodiments, the stem cellexpander is(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,which is present in the cell culture medium at a concentration of about0.75 micromolar (uM). In embodiments, the stem cell expander is(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,which is present in the cell culture medium at a concentration of about0.5 micromolar (uM). In embodiments of any of the foregoing, the cellculture medium additionally comprises compound 1.

In embodiments, the stem cell expander is a mixture of compound 1 and(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol.

In embodiments, the cells of the invention are contacted with one ormore stem cell expander molecules for a sufficient time and in asufficient amount to cause a 2 to 10,000-fold expansion of CD34+ cells,e.g., a 2-1000-fold expansion of CD34+ cells, e.g., a 2-100-foldexpansion of CD34+ cells, e.g., a 20-200-fold expansion of CD34+ cells.As described herein, the contacting with the one or more stem cellexpanders may be before the cells are contacted with a CRISPR system,e.g., as described herein, after the cells are contacted with a CRISPRsystem, e.g., as described herein, or a combination thereof. In anembodiment, the cells are contacted with one or more stem cell expandermolecules, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,for a sufficient time and in a sufficient amount to cause at least a2-fold expansion of CD34+ cells, e.g., CD34+ cells comprising an indelat or near the target site having complementarity to the targetingdomain of the gRNA of the CRISPR/Cas9 system introduced into said cell.In an embodiment, the cells are contacted with one or more stem cellexpander molecules, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,for a sufficient time and in a sufficient amount to cause at least a4-fold expansion of CD34+ cells, e.g., CD34+ cells comprising an indelat or near the target site having complementarity to the targetingdomain of the gRNA of the CRISPR/Cas9 system introduced into said cell.In an embodiment, the cells are contacted with one or more stem cellexpander molecules, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,for a sufficient time and in a sufficient amount to cause at least a5-fold expansion of CD34+ cells, e.g., CD34+ cells comprising an indelat or near the target site having complementarity to the targetingdomain of the gRNA of the CRISPR/Cas9 system introduced into said cell.In an embodiment, the cells are contacted with one or more stem cellexpander molecules for a sufficient time and in a sufficient amount tocause at least a 10-fold expansion of CD34+ cells. In an embodiment, thecells are contacted with one or more stem cell expander molecules for asufficient time and in a sufficient amount to cause at least a 20-foldexpansion of CD34+ cells. In an embodiment, the cells are contacted withone or more stem cell expander molecules for a sufficient time and in asufficient amount to cause at least a 30-fold expansion of CD34+ cells.In an embodiment, the cells are contacted with one or more stem cellexpander molecules for a sufficient time and in a sufficient amount tocause at least a 40-fold expansion of CD34+ cells. In an embodiment, thecells are contacted with one or more stem cell expander molecules for asufficient time and in a sufficient amount to cause at least a 50-foldexpansion of CD34+ cells. In an embodiment, the cells are contacted withone or more stem cell expander molecules for a sufficient time and in asufficient amount to cause at least a 60-fold expansion of CD34+ cells.In embodiments, the cells are contacted with the one or more stem cellexpanders for a period of about 1-60 days, e.g., about 1-50 days, e.g.,about 1-40 days, e.g., about 1-30 days, e.g., 1-20 days, e.g., about1-10 days, e.g., about 7 days, e.g., about 1-5 days, e.g., about 2-5days, e.g., about 2-4 days, e.g., about 2 days or, e.g., about 4 days.

In embodiments, the cells, e.g., HSPCs, e.g., as described herein, arecultured ex vivo for a period of about 1 hour to about 10 days, e.g., aperiod of about 12 hours to about 5 days, e.g., a period of about 12hours to 4 days, e.g., a period of about 1 day to about 4 days, e.g., aperiod of about 1 day to about 2 days, e.g., a period of about 1 day ora period of about 2 days, prior to the step of contacting the cells witha CRISPR system, e.g., described herein. In embodiments, said culturingprior to said contacting step is in a composition (e.g., a cell culturemedium) comprising a stem cell expander, e.g., described herein, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-olat a concentration of about 0.25 uM to about 1 uM, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-olat a concentration of about 0.75-0.5 micromolar. In embodiments, thecells are cultured ex vivo for a period of no more than about about 1day, e.g., no more than about 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,7, 6, 5, 4, 3, 2, or 1 hour(s) after the step of contacting the cellswith a CRISPR system, e.g., described herein, e.g., in a cell culturemedium which comprises a stem cell expander, e.g., described herein,e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-olat a concentration of about 0.25 uM to about 1 uM, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-olat a concentration of about 0.75-0.5 micromolar. In other embodiments,the cells are cultured ex vivo for a period of about 1 hour to about 14days, e.g., a period of about 12 hours to about 10 days, e.g., a periodof about 1 day to about 10 days, e.g., a period of about 1 day to about5 days, e.g., a period of about 1 day to about 4 days, e.g., a period ofabout 2 days to about 4 days, e.g., a period of about 2 days, about 3days or about 4 days, after the step of contacting the cells with aCRISPR system, e.g., described herein, in a cell culture medium, e.g.,which comprises a stem cell expander, e.g., described herein, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-olat a concentration of about 0.25 uM to about 1 uM, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-olat a concentration of about 0.75-0.5 micromolar.

In embodiments, the cell culture medium is a chemically defined medium.In embodiments, the cell culture medium may additionally contain, forexample, StemSpan SFEM (StemCell Technologies; Cat no. 09650). Inembodiments, the cell culture medium may alternatively or additionallycontain, for example, HSC Brew, GMP (Miltenyi). In embodiments, the cellculture media is serum free. In embodiments, the media may besupplemented with thrombopoietin (TPO), human Flt3 ligand (Flt-3L),human stem cell factor (SCF), human interleukin-6, L-glutamine, and/orpenicillin/streptomycin. In embodiments, the media is supplemented withthrombopoietin (TPO), human Flt3 ligand (Flt-3L), human stem cell factor(SCF), human interleukin-6, and L-glutamine. In other embodiments, themedia is supplemented with thrombopoietin (TPO), human Flt3 ligand(Flt-3L), human stem cell factor (SCF), and human interleukin-6. Inother embodiments the media is supplemented with thrombopoietin (TPO),human Flt3 ligand (Flt-3L), and human stem cell factor (SCF), but nothuman interleukin-6. In other embodiments, the media is supplementedwith human Flt3 ligand (Flt-3L), human stem cell factor (SCF), but nothuman thrombopoietin (TPO) or human interleukin-6. When present in themedium, the thrombopoietin (TPO), human Flt3 ligand (Flt-3L), human stemcell factor (SCF), human interleukin-6, and/or L-glutamine are eachpresent in a concentration ranging from about 1 ng/mL to about 1000ng/mL, e.g., a concentration ranging from about 10 ng/mL to about 500ng/mL, e.g., a concentration ranging from about 10 ng/mL to about 100ng/mL, e.g., a concentration ranging from about 25 ng/mL to about 75ng/mL, e.g., a concentration of about 50 ng/mL. In embodiments, each ofthe supplemented components is at the same concentration. In otherembodiments, each of the supplemented components is at a differentconcentration. In an embodiment, the medium comprises StemSpan SFEM(StemCell Technologies; Cat no. 09650), 50 ng/mL of thrombopoietin(Tpo), 50 ng/mL of human Flt3 ligand (Flt-3L), 50 ng/mL of human stemcell factor (SCF), and 50 ng/mL of human interleukin-6 (IL-6). In anembodiment, the medium comprises StemSpan SFEM (StemCell Technologies;Cat no. 09650), 50 ng/mL of thrombopoietin (Tpo), 50 ng/mL of human Flt3ligand (Flt-3L), and 50 ng/mL of human stem cell factor (SCF), and doesnot comprise IL-6. In embodiments, the media further comprises a stemcell expander, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-olat a concentration of 0.75 μM. In embodiments, the media furthercomprises a stem cell expander, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol,e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-olat a concentration of 0.5 μM. In embodiments, the media furthercomprises 1% L-glutamine and 2% penicillin/streptomycin. In embodiments,the cell culture medium is serum free.

XII. Combination Therapy

The present disclosure contemplates the use of the gRNA moleculesdescribed herein, or cells (e.g., hematopoietic stem cells, e.g., CD34+cells) modified with the gRNA molecules described herein, in combinationwith one or more other therapeutic modalities and/or agents. Thus, inaddition to the use of the gRNA molecules or cells modified with thegRNA molecules described herein, one may also administer to the subjectone or more “standard” therapies for treating hemoglobinopathies.

The one or more additional therapies for treating hemoglobinopathies mayinclude, for example, additional stem cell transplantation, e.g.,hematopoietic stem cell transplantation. The stem cell transplantationmay be allogeneic or autologous.

The one or more additional therapies for treating hemoglobinopathies mayinclude, for example, blood transfusion and/or iron chelation (e.g.,removal) therapy. Known iron chelation agents include, for example,deferoxamine and deferasirox.

The one or more additional therapies for treating hemoglobinopathies mayinclude, for example, folic acid supplements, or hydroxyurea (e.g.,5-hydroxyurea). The one or more additional therapies for treatinghemoglobinopathies may be hydroxyurea. In embodiments, the hydroxyureamay be administered at a dose of, for example, 10-35 mg/kg per day,e.g., 10-20 mg/kg per day. In embodiments, the hydroxyurea isadminstered at a dose of 10 mg/kg per day. In embodiments, thehydroxyurea is adminstered at a dose of 10 mg/kg per day. Inembodiments, the hydroxyurea is adminstered at a dose of 20 mg/kg perday. In embodiments, the hydroxyurea is administered before and/or afterthe cell (or population of cells), e.g., CD34+ cell (or population ofcells) of the invention, e.g., as described herein.

The one or more additional therapeutic agents may include, for example,an anti-p-selectin antibody, e.g., SelG1 (Selexys). P-selectinantibodies are described in, for example, PCT publication WO1993/021956,PCT publication WO1995/034324, PCT publication WO2005/100402, PCTpublication WO2008/069999, US patent application publicationUS2011/0293617, U.S. Pat. Nos. 5,800,815, 6,667,036, 8,945,565,8,377,440 and 9,068,001, the contents of each of which are incorporatedherein in their entirety.

The one or more additional agents may include, for example, a smallmolecule which upregulates fetal hemoglobin. Examples of such moleculesinclude TN1 (e.g., as described in Nam, T. et al., ChemMedChem 2011, 6,777-780, DOI: 10.1002/cmdc.201000505, herein incorporated by reference).

The one or more additional therapies may also include irradiation orother bone marrow ablation therapies known in the art. An example ofsuch a therapy is busulfan. Such additional therapy may be performedprior to introduction of the cells of the invention into the subject. Inan embodiment the methods of treatment described herein (e.g., themethods of treatment that include administration of cells (e.g., HSPCs)modified by the methods described herein (e.g., modified with a CRISPRsystem described herein, e.g., to increase HbF production)), the methoddoes not include the step of bone marrow ablation. In embodiments, themethods include a partial bone marrow ablation step.

The therapies described herein (e.g., comprising administering apopulation of HSPCs, e.g., HSPCs modified using a CRISPR systemdescribed herein) may also be combined with an additional therapeuticagent. In an embodiment, the additional therapeutic agent is an HDACinhibitor, e.g., panobinostat. In an embodiment, the additionaltherapeutic is a compound described in PCT Publication No.WO2014/150256, e.g., a compound described in Table 1 of WO2014/150256,e.g., GBT440. Other examples of HDAC inhibitors include, for example,suberoylanilide hydroxamic acid (SAHA). The one or more additionalagents may include, for example, a DNA methylation inhibitor.

Such agents have been shown to increase the HbF induction in cellshaving reduced BCL11a activity (e.g., Jian Xu et al, Science 334, 993(2011); DOI: 0.1126/science.1211053, herein incorporated by reference).Other HDAC inhibitors include any HDAC inhibitor known in the art, forexample, trichostatin A, HC toxin, DACI-2, FK228, DACI-14, depudicin,DACI-16, tubacin, NK57, MAZ1536, NK125, Scriptaid, Pyroxamide, MS-275,ITF-2357, MCG-D0103, CRA-024781, CI-994, and LBH589 (see, e.g., BradnerJ E, et al., PNAS, 2010 (vol. 107:28), 12617-12622, herein incorporatedby reference in its entirety).

The gRNA molecules described herein, or cells (e.g., hematopoietic stemcells, e.g., CD34+ cells) modified with the gRNA molecules describedherein, and the co-therapeutic agent or co-therapy can be administeredin the same formulation or separately. In the case of separateadministration, the gRNA molecules described herein, or cells modifiedwith the gRNA molecules described herein, can be administered before,after or concurrently with the co-therapeutic or co-therapy. One agentmay precede or follow administration of the other agent by intervalsranging from minutes to weeks. In embodiments where two or moredifferent kinds of therapeutic agents are applied separately to asubject, one would generally ensure that a significant period of timedid not expire between the time of each delivery, such that thesedifferent kinds of agents would still be able to exert an advantageouslycombined effect on the target tissues or cells.

XIII. Modified Nucleosides, Nucleotides, and Nucleic Acids

Modified nucleosides and modified nucleotides can be present in nucleicacids, e.g., particularly gRNA, but also other forms of RNA, e.g., mRNA,RNAi, or siRNA. As described herein “nucleoside” is defined as acompound containing a five-carbon sugar molecule (a pentose or ribose)or derivative thereof, and an organic base, purine or pyrimidine, or aderivative thereof. As described herein, “nucleotide” is defined as anucleoside further comprising a phosphate group.

Modified nucleosides and nucleotides can include one or more of:

(i) alteration, e.g., replacement, of one or both of the non-linkingphosphate oxygens and/or of one or more of the linking phosphate oxygensin the phosphodiester backbone linkage;

(ii) alteration, e.g., replacement, of a constituent of the ribosesugar, e.g., of the 2′ hydroxyl on the ribose sugar;

(iii) wholesale replacement of the phosphate moiety with “dephospho”linkers;

(iv) modification or replacement of a naturally occurring nucleobase,including with a non-canonical nucleobase;

(v) replacement or modification of the ribose-phosphate backbone;

(vi) modification of the 3′ end or 5′ end of the oligonucleotide, e.g.,removal, modification or replacement of a terminal phosphate group orconjugation of a moiety, cap or linker; and

(vii) modification or replacement of the sugar.

The modifications listed above can be combined to provide modifiednucleosides and nucleotides that can have two, three, four, or moremodifications. For example, a modified nucleoside or nucleotide can havea modified sugar and a modified nucleobase. In an embodiment, every baseof a gRNA is modified, e.g., all bases have a modified phosphate group,e.g., all are phosphorothioate groups. In an embodiment, all, orsubstantially all, of the phosphate groups of a unimolecular or modulargRNA molecule are replaced with phosphorothioate groups. In embodiments,one or more of the five 3′-terminal bases and/or one or more of the five5′-terminal bases of the gRNA are modified with a phosphorothioategroup.

In an embodiment, modified nucleotides, e.g., nucleotides havingmodifications as described herein, can be incorporated into a nucleicacid, e.g., a “modified nucleic acid.” In some embodiments, the modifiednucleic acids comprise one, two, three or more modified nucleotides. Insome embodiments, at least 5% (e.g., at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, orabout 100%) of the positions in a modified nucleic acid are a modifiednucleotides.

Unmodified nucleic acids can be prone to degradation by, e.g., cellularnucleases. For example, nucleases can hydrolyze nucleic acidphosphodiester bonds. Accordingly, in one aspect the modified nucleicacids described herein can contain one or more modified nucleosides ornucleotides, e.g., to introduce stability toward nucleases.

In some embodiments, the modified nucleosides, modified nucleotides, andmodified nucleic acids described herein can exhibit a reduced innateimmune response when introduced into a population of cells, both in vivoand ex vivo. The term “innate immune response” includes a cellularresponse to exogenous nucleic acids, including single stranded nucleicacids, generally of viral or bacterial origin, which involves theinduction of cytokine expression and release, particularly theinterferons, and cell death. In some embodiments, the modifiednucleosides, modified nucleotides, and modified nucleic acids describedherein can disrupt binding of a major groove interacting partner withthe nucleic acid.

In some embodiments, the modified nucleosides, modified nucleotides, andmodified nucleic acids described herein can exhibit a reduced innateimmune response when introduced into a population of cells, both in vivoand ex vivo, and also disrupt binding of a major groove interactingpartner with the nucleic acid.

Definitions of Chemical Groups

As used herein, “alkyl” is meant to refer to a saturated hydrocarbongroup which is straight-chained or branched. Example alkyl groupsinclude methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl),butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl,isopentyl, neopentyl), and the like. An alkyl group can contain from 1to about 20, from 2 to about 20, from 1 to about 12, from 1 to about 8,from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms.

As used herein, “aryl” refers to monocyclic or polycyclic (e.g., having2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example,phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and thelike. In some embodiments, aryl groups have from 6 to about 20 carbonatoms.

As used herein, “alkenyl” refers to an aliphatic group containing atleast one double bond. As used herein, “alkynyl” refers to a straight orbranched hydrocarbon chain containing 2-12 carbon atoms andcharacterized in having one or more triple bonds. Examples of alkynylgroups include, but are not limited to, ethynyl, propargyl, and3-hexynyl.

As used herein, “arylalkyl” or “aralkyl” refers to an alkyl moiety inwhich an alkyl hydrogen atom is replaced by an aryl group. Aralkylincludes groups in which more than one hydrogen atom has been replacedby an aryl group. Examples of “arylalkyl” or “aralkyl” include benzyl,2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and tritylgroups.

As used herein, “cycloalkyl” refers to a cyclic, bicyclic, tricyclic, orpolycyclic non-aromatic hydrocarbon groups having 3 to 12 carbons.Examples of cycloalkyl moieties include, but are not limited to,cyclopropyl, cyclopentyl, and cyclohexyl.

As used herein, “heterocyclyl” refers to a monovalent radical of aheterocyclic ring system. Representative heterocyclyls include, withoutlimitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl,pyrrolidonyl, piperidinyl, pyrrolinyl, piperazinyl, dioxanyl,dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, and morpholinyl.

As used herein, “heteroaryl” refers to a monovalent radical of aheteroaromatic ring system. Examples of heteroaryl moieties include, butare not limited to, imidazolyl, oxazolyl, thiazolyl, triazolyl,pyrrolyl, furanyl, indolyl, thiophenyl pyrazolyl, pyridinyl, pyrazinyl,pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl,quinolyl, and pteridinyl.

Phosphate Backbone Modifications

The Phosphate Group

In some embodiments, the phosphate group of a modified nucleotide can bemodified by replacing one or more of the oxygens with a differentsubstituent. Further, the modified nucleotide, e.g., modified nucleotidepresent in a modified nucleic acid, can include the wholesalereplacement of an unmodified phosphate moiety with a modified phosphateas described herein. In some embodiments, the modification of thephosphate backbone can include alterations that result in either anuncharged linker or a charged linker with unsymmetrical chargedistribution.

Examples of modified phosphate groups include, phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidites, alkyl or aryl phosphonates andphosphotriesters. In some embodiments, one of the non-bridging phosphateoxygen atoms in the phosphate backbone moiety can be replaced by any ofthe following groups: sulfur (S), selenium (Se), BR₃ (wherein R can be,e.g., hydrogen, alkyl, or aryl), C (e.g., an alkyl group, an aryl group,and the like), H, NR₂ (wherein R can be, e.g., hydrogen, alkyl, oraryl), or OR (wherein R can be, e.g., alkyl or aryl). The phosphorousatom in an unmodified phosphate group is achiral. However, replacementof one of the non-bridging oxygens with one of the above atoms or groupsof atoms can render the phosphorous atom chiral; that is to say that aphosphorous atom in a phosphate group modified in this way is astereogenic center. The stereogenic phosphorous atom can possess eitherthe “R” configuration (herein Rp) or the “S” configuration (herein Sp).

Phosphorodithioates have both non-bridging oxygens replaced by sulfur.The phosphorus center in the phosphorodithioates is achiral whichprecludes the formation of oligoribonucleotide diastereomers. In someembodiments, modifications to one or both non-bridging oxygens can alsoinclude the replacement of the non-bridging oxygens with a groupindependently selected from S, Se, B, C, H, N, and OR (R can be, e.g.,alkyl or aryl).

The phosphate linker can also be modified by replacement of a bridgingoxygen, (i.e., the oxygen that links the phosphate to the nucleoside),with nitrogen (bridged phosphoroamidites), sulfur (bridgedphosphorothioates) and carbon (bridged methylenephosphonates). Thereplacement can occur at either linking oxygen or at both of the linkingoxygens.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containingconnectors. In some embodiments, the charge phosphate group can bereplaced by a neutral moiety.

Examples of moieties which can replace the phosphate group can include,without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane,carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxidelinker, sulfonate, sulfonamide, thioformacetal, formacetyl, oxime,methyleneimino, methylenemethylimino, methylenehydrazo,methylenedimethylhydrazo and methyleneoxymethylimino.

Replacement of the Ribophosphate Backbone

Scaffolds that can mimic nucleic acids can also be constructed whereinthe phosphate linker and ribose sugar are replaced by nuclease resistantnucleoside or nucleotide surrogates. In some embodiments, thenucleobases can be tethered by a surrogate backbone. Examples caninclude, without limitation, the morpholino, cyclobutyl, pyrrolidine andpeptide nucleic acid (PNA) nucleoside surrogates.

Sugar Modifications

The modified nucleosides and modified nucleotides can include one ormore modifications to the sugar group. For example, the 2′ hydroxylgroup (OH) can be modified or replaced with a number of different “oxy”or “deoxy” substituents. In some embodiments, modifications to the 2′hydroxyl group can enhance the stability of the nucleic acid since thehydroxyl can no longer be deprotonated to form a 2′-alkoxide ion. The2′-alkoxide can catalyze degradation by intramolecular nucleophilicattack on the linker phosphorus atom.

Examples of “oxy”-2′ hydroxyl group modifications can include alkoxy oraryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl,heteroaryl or a sugar); polyethyleneglycols (PEG),0(CH₂CH₂0)_(n)CH₂CH₂OR wherein R can be, e.g., H or optionallysubstituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8,from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4to 16, and from 4 to 20). In some embodiments, the “oxy”-2′ hydroxylgroup modification can include “locked” nucleic acids (LNA) in which the2′ hydroxyl can be connected, e.g., by a Ci-₆ alkylene or Cj-6heteroalkylene bridge, to the 4′ carbon of the same ribose sugar, whereexemplary bridges can include methylene, propylene, ether, or aminobridges; O-amino (wherein amino can be, e.g., NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, ordiheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy,0(CH₂)_(n)-amino, (wherein amino can be, e.g., NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, ordiheteroarylamino, ethylenediamine, or polyamino). In some embodiments,the “oxy”-2′ hydroxyl group modification can include the methoxyethylgroup (MOE), (OCH₂CH₂OCH₃, e.g., a PEG derivative).

“Deoxy” modifications can include hydrogen (i.e. deoxyribose sugars,e.g., at the overhang portions of partially ds RNA); halo (e.g., bromo,chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino,heteroarylamino, diheteroarylamino, or amino acid);NH(CH₂CH₂NH)_(n)CH2CH₂— amino (wherein amino can be, e.g., as describedherein), —NHC(0)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl,aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which maybe optionally substituted with e.g., an amino as described herein.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified nucleic acid can include nucleotidescontaining e.g., arabinose, as the sugar. The nucleotide “monomer” canhave an alpha linkage at the Γ position on the sugar, e.g.,alpha-nucleosides. The modified nucleic acids can also include “abasic”sugars, which lack a nucleobase at C—. These abasic sugars can also befurther modified at one or more of the constituent sugar atoms. Themodified nucleic acids can also include one or more sugars that are inthe L form, e.g. L-nucleosides.

Generally, RNA includes the sugar group ribose, which is a 5-memberedring having an oxygen. Exemplary modified nucleosides and modifiednucleotides can include, without limitation, replacement of the oxygenin ribose (e.g., with sulfur (S), selenium (Se), or alkylene, such as,e.g., methylene or ethylene); addition of a double bond (e.g., toreplace ribose with cyclopentenyl or cyclohexenyl); ring contraction ofribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ringexpansion of ribose (e.g., to form a 6- or 7-membered ring having anadditional carbon or heteroatom, such as for example, anhydrohexitol,altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that alsohas a phosphoramidate backbone). In some embodiments, the modifiednucleotides can include multicyclic forms (e.g., tricyclo; and“unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA orS-GNA, where ribose is replaced by glycol units attached tophosphodiester bonds), threose nucleic acid (TNA, where ribose isreplaced with a-L-threofuranosyl-(3′-→2′)).

Modifications on the Nucleobase

The modified nucleosides and modified nucleotides described herein,which can be incorporated into a modified nucleic acid, can include amodified nucleobase. Examples of nucleobases include, but are notlimited to, adenine (A), guanine (G), cytosine (C), and uracil (U).These nucleobases can be modified or wholly replaced to provide modifiednucleosides and modified nucleotides that can be incorporated intomodified nucleic acids. The nucleobase of the nucleotide can beindependently selected from a purine, a pyrimidine, a purine orpyrimidine analog. In some embodiments, the nucleobase can include, forexample, naturally-occurring and synthetic derivatives of a base.

Uracil

In some embodiments, the modified nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having a modified uracil includewithout limitation pseudouridine (ψ), pyridin-4-one ribonucleoside,5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine(s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m³U),5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine5-oxyacetic acid methyl ester (mcmo{circumflex over ( )}U),5-carboxymethyl-uridine (cm⁵U), 1-carboxymethyl-pseudouridine,5-carboxyhydroxymethyl-uridine (chm⁵U), 5-carboxyhydroxymethyl-uridinemethyl ester (mchm⁵U), 5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s2U),5-aminomethyl-2-thio-uridine (nm⁵s2U), 5-methylaminomethyl-uridine(mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s2U),5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm \s2U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(xcm⁵U), 1-taurinomethyl-pseudouridine,5-taurinomethyl-2-thio-uridine(Trn⁵s2U),1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U, i.e.,having the nucleobase deoxythymine), 1-methyl-pseudouridine (ιτι′ψ).5-methyl-2-thio-uridine (m⁵s2U), l-methyl-4-thio-pseudouridine (m's\|/), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m′V),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydroundine (D),dihydropseudoundine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropy pseudouridine5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyp-2-thio-uridine (inm⁵s2U), a-thio-uridine,2′-0-methyl-uridine (Urn), 5,2′-0-dimethyl-uridine (m⁵Um),2′-0-methyl-pseudouridine (ψπι), 2-thio-2′-0-methyl-uridine (s2Um),5-methoxycarbonylmethyl-2′-0-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-0-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-0-methyl-uridine (cmnm⁵Um),3,2′-0-dimethyl-uridine (m³Um),5-(isopentenylaminomethyl)-2′-0-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl) uridine, 5-[3-(1-E-propenylamino)uridine,pyrazolo[3,4-d]pyrimidines, xanthine, and hypoxanthine.

Cytosine

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine includewithout limitation 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine,3-methyl-cytidine (m³C), N4-acetyl-cytidine (act), 5-formyl-cytidine(f⁵C), N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C),5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine(hm⁵C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine,pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C),2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k²C), a-thio-cytidine, 2′-0-methyl-cytidine (Cm),5,2′-0-dimethyl-cytidine (m⁵Cm), N4-acetyl-2′-0-methyl-cytidine (ac⁴Cm),N4,2′-0-dimethyl-cytidine (m⁴Cm), 5-formyl-2′-0-methyl-cytidine (f⁵Cm),N4,N4,2′-0-trimethyl-cytidine (m⁴ ₂Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

Adenine

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine includewithout limitation 2-amino-purine, 2,6-diaminopurine,2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine(e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine,7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m′A),2-methyl-adenine (m A), N6-methyl-adenosine (m⁶A),2-methylthio-N6-methyl-adenosine (ms2 m⁶A), N6-isopentenyl-adenosine(i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A),N6-(cis-hydroxyisopentanyl)adenosine (io⁶A),2-methylthio-N6-(cis-hydroxyisopentanyl)adenosine (ms2io⁶A),N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine(t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A),2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g⁶A),N6,N6-dimethyl-adenosine (m⁶ ₂A), N6-hydroxynorvalylcarbamoyl-adenosine(hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn⁶A),N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, a-thio-adenosine, 2′-0-methyl-adenosine (Am),N⁶,2′-0-dimethyl-adenosine (m⁵Am), N⁶-Methyl-2′-deoxyadenosine,N6,N6,2′-0-trimethyl-adenosine (m⁶ ₂Am), 1,2′-0-dimethyl-adenosine (m′Am), 2′-0-ribosyladenosine (phosphate) (Ar(p)),2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine,2′-F-ara-adenosine, 2′-F-adenosine, 2′-OH-ara-adenosine, andN6-(19-amino-pentaoxanonadecyl)-adenosine.

Guanine

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includewithout limitation inosine (I), 1-methyl-inosine (m′l), wyosine (imG),methylwyosine (mimG), 4-demethyl-wyo″sine (imG-14), isowyosine (imG2),wybutosine (yW), peroxywybutosine (o₂yW), hydroxywybutosine (OHyW),undemriodified hydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine(Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ),mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ₀),7-aminomethyl-7-deaza-guanosine (preQi), archaeosine (G),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methyl-guanosine (m′G), N2-methyl-guanosine (m²G),N2,N2-dimethyl-guanosine (m² ₂G), N2,7-dimethyl-guanosine (m²,7G), N2,N2,7-dimethyl-guanosine (m²,2,7G), 8-oxo-guanosine,7-methyl-8-oxo-guanosine, 1-meth thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine,a-thio-guanosine, 2′-0-methyl-guanosine (Gm),N2-methyl-2′-0-methyl-guanosine (m¾m),N2,N2-dimethyl-2′-0-methyl-guanosine (m² ₂Gm),1-methyl-2′-0-methyl-guanosine (m'Gm),N2,7-dimethyl-2′-0-methyl-guanosine (m²,7Gm), 2′-0-methyl-inosine (Im),1,2′-0-dimethyl-inosine (m'lm), 0⁶-phenyl-2′-deoxyinosine,2′-0-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine,0⁶-methy]-guanosine, 0⁶-Methyl-2′-deoxyguanosine, 2′-F-ara-guanosine,and 2′-F-guanosine.

Modified gRNAs

In some embodiments, the modified nucleic acids can be modified gRNAs.In some embodiments, gRNAs can be modified at the 3′ end. In thisembodiment, the gRNAs can be modified at the 3′ terminal U ribose. Forexample, the two terminal hydroxyl groups of the U ribose can beoxidized to aldehyde groups and a concomitant opening of the ribose ringto afford a modified nucleoside, wherein U can be an unmodified ormodified uridine.

In another embodiment, the 3′ terminal U can be modified with a 2′ 3′cyclic phosphate, wherein U can be an unmodified or modified uridine. Insome embodiments, the gRNA molecules may contain 3′ nucleotides whichcan be stabilized against degradation, e.g., by incorporating one ormore of the modified nucleotides described herein. In this embodiment,e.g., uridines can be replaced with modified uridines, e.g.,5-(2-amino)propyl uridine, and 5-bromo uridine, or with any of themodified uridines described herein; adenosines and guanosines can bereplaced with modified adenosines and guanosines, e.g., withmodifications at the 8-position, e.g., 8-bromo guanosine, or with any ofthe modified adenosines or guanosines described herein. In someembodiments, deaza nucleotides, e.g., 7-deaza-adenosine, can beincorporated into the gRNA. In some embodiments, 0- and N-alkylatednucleotides, e.g., N6-methyl adenosine, can be incorporated into thegRNA. In some embodiments, sugar-modified ribonucleotides can beincorporated, e.g., wherein the 2′ OH— group is replaced by a groupselected from H, —OR, —R (wherein R can be, e.g., methyl, alkyl,cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, —SH, —SR (whereinR can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar),amino (wherein amino can be, e.g., NH₂; alkylamino, dialkylamino,heterocyclyl, acylamino, diarylamino, heteroarylamino,diheteroarylamino, or amino acid); or cyano (—CN). In some embodiments,the phosphate backbone can be modified as described herein, e.g., with aphosphothioate group. In some embodiments, the nucleotides in theoverhang region of the gRNA can each independently be a modified orunmodified nucleotide including, but not limited to 2′-sugar modified,such as, 2-F 2′-0-methyl, thymidine (T),2′-O-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine(Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinationsthereof.

In an embodiment, a one or more or all of the nucleotides in singlestranded overhang of an RNA molecule, e.g., a gRNA molecule, aredeoxynucleotides.

Pharmaceutical Compositions

Pharmaceutical compositions of the present invention may comprise a gRNAmolecule described herein, e.g., a plurality of gRNA molecules asdescribed herein, or a cell (e.g., a population of cells, e.g., apopulation of hematopoietic stem cells, e.g., of CD34+ cells) comprisingone or more cells modified with one or more gRNA molecules describedherein, in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.Compositions of the present invention are in one aspect formulated forintravenous administration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

In one embodiment, the pharmaceutical composition is substantially freeof, e.g., there are no detectable levels of a contaminant, e.g.,selected from the group consisting of endotoxin, mycoplasma, mouseantibodies, pooled human serum, bovine serum albumin, bovine serum,culture media components, unwanted CRISPR system components, a bacteriumand a fungus. In one embodiment, the bacterium is at least one selectedfrom the group consisting of Alcaligenes faecalis, Candida albicans,Escherichia coli, Haemophilus influenza, Neisseria meningitides,Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia,and Streptococcus pyogenes group A.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patienttransarterially, subcutaneously, intradermally, intratumorally,intranodally, intramedullary, intramuscularly, by intravenous (i.v.)injection, or intraperitoneally. In one aspect, the compositions of thepresent invention are administered to a patient by intradermal orsubcutaneous injection. In one aspect, the cell compositions of thepresent invention are administered by i.v. injection.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices.

Cells

The invention also relates to cells comprising a gRNA molecule of theinvention, or nucleic acid encoding said gRNA molecules.

In an aspect the cells are cells made by a process described herein.

In embodiments, the cells are hematopoietic stem cells (e.g.,hematopoietic stem and progenitor cells; HSPCs), for example, CD34+ stemcells. In embodiments, the cells are CD34+/CD90+ stem cells. Inembodiments, the cells are CD34+/CD90− stem cells. In embodiments, thecells are human hematopoietic stem cells. In embodiments, the cells areautologous. In embodiments, the cells are allogeneic.

In embodiments, the cells are derived from bone marrow, e.g., autologousbone marrow. In embodiments, the cells are derived from peripheralblood, e.g., mobilized peripheral blood, e.g., autologous mobilizedperipheral blood. In embodiments employing mobilized peripheral blood,the cells are isolated from patients who have been administered amobilization agent. In embodiments, the mobilization agent is G-CSF. Inembodiments, the mobilization agent is Plerixafor® (AMD3100). Inembodiments, the mobilization agent comprises a combination of G-CSF andPlerixafor® (AMD3100)). In embodiments, the cells are derived fromumbilical cord blood, e.g., allogeneic umbilical cord blood. Inembodiments, the cells are derived from a hemoglobinopathy patient, forexample a patient with sickle cell disease or a patient with athalassemia, e.g., beta-thalassemia.

In embodiments, the cells are mammalian. In embodiments, the cells arehuman. In embodiments, the cells are derived from a hemoglobinopathypatient, for example a patient with sickle cell disease or a patientwith a thalassemia, e.g., beta-thalassemia.

In an aspect, the invention provides a cell comprising a modification oralteration, e.g., an indel, at or near (e.g., within 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides of) anucleic acid sequence having complementarity to a gRNA molecule or gRNAmolecules, e.g., as described herein, introduced into said cells, e.g.,as part of a CRISPR system as described herein. In embodiments, the cellis a CD34+ cell. In embodiments, the altered or modified cell, e.g.,CD34+ cell, maintains the ability to differentiate into cells ofmultiple lineages, e.g., maintains the ability to differentiate intocells of the erythroid lineage. In embodiments, the altered or modifiedcell, e.g., CD34+ cell, has undergone or is able to undergo at least 2,at least 3, at least 4, at least 5, at least 6, at least 7, at least 8,at least 9 or at least 10 or more doublings in culture, e.g., in culturecomprising a stem cell expander, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol.In embodiments, the altered or modified cell, e.g., CD34+ cell, hasundergone or is able to undergo at least 5, e.g., about 5, doublings inculture, e.g., in culture comprising a stem cell expander molecule,e.g., as described herein, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol.In embodiments the altered or modified cell, e.g., CD34+ cell, exhibitsand/or is able to differentiate into a cell, e.g., into a cell of theerythroid lineage, e.g., into a red blood cell, that exhibits increasedfetal hemoglobin level (e.g., expression level and/or protein level),e.g., at least a 20% increase in fetal hemoglobin protein level,relative to a similar unmodified or unaltered cell. In embodiments thealtered or modified cell, e.g., CD34+ cell, exhibits and/or is able todifferentiate into a cell, e.g., into a cell of the erythroid lineage,e.g., into a red blood cell, that exhibits increased fetal hemoglobinlevel (e.g., expression level and/or protein level), relative to asimilar unmodified or unaltered cell, e.g., produces at least 6picograms, e.g., at least 7 picograms, at least 8 picograms, at least 9picograms, or at least 10 picograms of fetal hemoglobin. In embodimentsthe altered or modified cell, e.g., CD34+ cell, exhibits and/or is ableto differentiate into a cell, e.g., into a cell of the erythroidlineage, e.g., into a red blood cell, that exhibits increased fetalhemoglobin level (e.g., expression level and/or protein level), relativeto a similar unmodified or unaltered cell, e.g., produces about 6 toabout 12, about 6 to about 7, about 7 to about 8, about 8 to about 9,about 9 to about 10, about 10 to about 11 or about 11 to about 12picograms of fetal hemoglobin.

In an aspect, the invention provides a population of cells comprisingcells having a modification or alteration, e.g., an indel, at or near(e.g., within 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2 or 1 nucleotides of) a nucleic acid sequence havingcomplementarity to a gRNA molecule or gRNA molecules, e.g., as describedherein, introduced into said cells, e.g., as part of a CRISPR system asdescribed herein. In embodiments, at least 50%, e.g., at least 60%, atleast 70%, at least 80% or at least 90% of the cells of the populationhave the modification or alteration (e.g., have at least onemodification or alteration), e.g., as measured by NGS, e.g., asdescribed herein, e.g., at day two following introduction of the gRNAand/or CRISPR system of the invention. In embodiments, at least 90%,e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% of thecells of the population have the modification or alteration (e.g., haveat least one modification or alteration), e.g., as measured by NGS,e.g., as described herein, e.g., at day two following introduction ofthe gRNA and/or CRISPR system of the invention. In embodiments, thepopulation of cells comprise CD34+ cells, e.g., comprise at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 95% or at least about 98% CD34+ cells.In embodiments, the population of cells comprising the altered ormodified cells, e.g., CD34+ cells, maintain the ability to produce,e.g., differentiate into, cells of multiple lineages, e.g., maintainsthe ability to produce, e.g., differentiate into, cells of the erythroidlineage. In embodiments, the population of cells, e.g., population ofCD34+ cells, has undergone or is able to undergo at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9or at least 10 or more population doublings in culture, e.g., in culturecomprising a stem cell expander, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol.In embodiments, the population of altered or modified cells, e.g.,population of CD34+ cells, has undergone or is capable of undergoing atleast 5, e.g., about 5, population doublings in culture, e.g., inculture comprising a stem cell expander molecule, e.g., as describedherein, e.g.,(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol.In embodiments the population of cells comprising altered or modifiedcells, e.g., CD34+ cells, exhibits and/or is able to differentiate intoa population of cells, e.g., into a population of cells of the erythroidlineage, e.g., into a population of red blood cells, that exhibitsincreased fetal hemoglobin level (e.g., expression level and/or proteinlevel), e.g., at least a 20% increase in fetal hemoglobin protein level,relative to a similar unmodified or unaltered cells. In embodiments thepopulation of cells comprising altered or modified cells, e.g., CD34+cells, exhibits and/or is able to differentiate into a population ofcells, e.g., into a population of cells of the erythroid lineage, e.g.,into a population of red blood cells, that exhibits increased fetalhemoglobin level (e.g., expression level and/or protein level), relativeto a similar unmodified or unaltered cells, e.g., comprises cells thatproduce at least 6 picograms, e.g., at least 7 picograms, at least 8picograms, at least 9 picograms, or at least 10 picograms of fetalhemoglobin per cell. In embodiments the population of altered ormodified cells, e.g., CD34+ cells, exhibits and/or is able todifferentiate into a population of cells, e.g., into a population ofcells of the erythroid lineage, e.g., into a population of red bloodcells, that exhibits increased fetal hemoglobin level (e.g., expressionlevel and/or protein level), relative to a similar unmodified orunaltered cell, e.g., comprises cells that produce about 6 to about 12,about 6 to about 7, about 7 to about 8, about 8 to about 9, about 9 toabout 10, about 10 to about 11 or about 11 to about 12 picograms offetal hemoglobin per cell.

In embodiments, the population of cells, e.g., as described herein,comprises at least about 1e3 cells. In embodiments, the population ofcells, e.g., as described herein, comprises at least about 1e4 cells. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 1e5 cells. In embodiments, the population ofcells, e.g., as described herein, comprises at least about 1e6 cells. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 1e7 cells. In embodiments, the population ofcells, e.g., as described herein, comprises at least about 1e8 cells. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 1e9 cells. In embodiments, the population ofcells, e.g., as described herein, comprises at least about 1e10 cells.In embodiments, the population of cells, e.g., as described herein,comprises at least about 1e11 cells. In embodiments, the population ofcells, e.g., as described herein, comprises at least about 1e12 cells.In embodiments, the population of cells, e.g., as described herein,comprises at least about 1e13 cells per kilogram body weight of thepatient to which they are to be administered. In embodiments, thepopulation of cells, e.g., as described herein, comprises at least about1e6 cells per kilogram body weight of the patient to which they are tobe administered. In embodiments, the population of cells, e.g., asdescribed herein, comprises at least about 2e6 cells per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 3e6 cells per kilogram body weight of thepatient to which they are to be administered. In embodiments, thepopulation of cells, e.g., as described herein, comprises at least about4e6 cells per kilogram body weight of the patient to which they are tobe administered. In embodiments, the population of cells, e.g., asdescribed herein, comprises at least about 5e6 cells per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 6e6 cells per kilogram body weight of thepatient to which they are to be administered. In embodiments, thepopulation of cells, e.g., as described herein, comprises at least about7e6 cells per kilogram body weight of the patient to which they are tobe administered. In embodiments, the population of cells, e.g., asdescribed herein, comprises at least about 8e6 cells per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 9e6 cells per kilogram body weight of thepatient to which they are to be administered. In embodiments, thepopulation of cells, e.g., as described herein, comprises at least about1e7 cells per kilogram body weight of the patient to which they are tobe administered. In embodiments, the population of cells, e.g., asdescribed herein, comprises at least about 2e7 cells per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 3e7 cells per kilogram body weight of thepatient to which they are to be administered. In embodiments, thepopulation of cells, e.g., as described herein, comprises at least about4e7 cells per kilogram body weight of the patient to which they are tobe administered. In embodiments, the population of cells, e.g., asdescribed herein, comprises at least about 5e7 cells per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 6e7 cells per kilogram body weight of thepatient to which they are to be administered. In embodiments, thepopulation of cells, e.g., as described herein, comprises at least about7e7 cells per kilogram body weight of the patient to which they are tobe administered. In embodiments, the population of cells, e.g., asdescribed herein, comprises at least about 8e7 cells per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 9e7 cells per kilogram body weight of thepatient to which they are to be administered. In embodiments, thepopulation of cells, e.g., as described herein, comprises at least about1e8 cells per kilogram body weight of the patient to which they are tobe administered. In any of the aforementioned embodiments, thepopulation of cells may comprise at least about 50% (for example, atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95% or at least about 99%) HSPCs, e.g., CD34+ cells.In any of the aforementioned embodiments, the population of cells maycomprise about 60% HSPCs, e.g., CD34+ cells. In an embodiment, thepopulation of cells, e.g., as described herein, comprises about 3e7cells and comprises about 2e7 HSPCs, e.g., CD34+ cells. As usedthroughout this application, the scientific notation [number]e[number]is given its ordinary meaning. Thus, for example, 2e6 is equivalent to2×10⁶ or 2,000,000.

In embodiments, the population of cells, e.g., as described herein,comprises at least about 1e6 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 1.5e6 HSPCs, e.g., CD34+ cells, per kilogrambody weight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 2e6 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 3e6 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 4e6 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 5e6 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 6e6 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 7e6 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 8e6 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 9e6 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 1e7 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 2e7 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 3e7 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 4e7 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 5e7 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 6e7 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 7e7 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 8e7 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 9e7 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 1e8 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 2e8 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 3e8 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 4e8 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered. Inembodiments, the population of cells, e.g., as described herein,comprises at least about 5e8 HSPCs, e.g., CD34+ cells, per kilogram bodyweight of the patient to which they are to be administered.

In embodiments, the population of cells, e.g., as described herein,comprises about 1e6 HSPCs, e.g., CD34+ cells, per kilogram body weightof the patient to which they are to be administered. In embodiments, thepopulation of cells, e.g., as described herein, comprises about 1.5e6HSPCs, e.g., CD34+ cells, per kilogram body weight of the patient towhich they are to be administered. In embodiments, the population ofcells, e.g., as described herein, comprises about 2e6 HSPCs, e.g., CD34+cells, per kilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 3e6 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 4e6 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 5e6 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 6e6 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 7e6 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 8e6 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 9e6 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 1e7 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 2e7 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 3e7 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 4e7 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 5e7 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 6e7 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 7e7 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 8e7 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 9e7 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 1e8 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 2e8 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 3e8 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 4e8 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises about 5e8 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered.

In embodiments, the population of cells, e.g., as described herein,comprises from about 2e6 to about 10e6 HSPCs, e.g., CD34+ cells, perkilogram body weight of the patient to which they are to beadministered. In embodiments, the population of cells, e.g., asdescribed herein, comprises from 2e6 to 10e6 HSPCs, e.g., CD34+ cells,per kilogram body weight of the patient to which they are to beadministered.

The cells of the invention may comprise a gRNA molecule of the presentinvention, or nucleic acid encoding said gRNA molecule, and a Cas9molecule of the present invention, or nucleic acid encoding said Cas9molecule. In an embodiment, the cells of the invention may comprise aribonuclear protein (RNP) complex which comprises a gRNA molecule of theinvention and a Cas9 molecule of the invention.

The cells of the invention are preferrably modified to comprise a gRNAmolecule of the invention ex vivo, for example by a method describedherein, e.g., by electroporation or by TRIAMF (as described in patentapplication PCT/US2017/54110, incorporated herein by reference in itsentirety).

The cells of the invention include cells in which expression of one ormore genes has been altered, for example, reduced or inhibited, byintroduction of a CRISPR system comprising a gRNA of the invention. Forexample, the cells of the present invention may have a reduced level ofbeta globin (e.g., hemoglobin beta comprising a sickling mutation)expression relative to unmodified cells. As another example, the cellsof the present invention may have an increased level of fetal hemoglobinexpression relative to unmodified cells. Alternatively, or in addition,a cell of the invention may give rise, e.g., differentiate into, anothertype of cell, e.g., an erythrocyte, that has an increased level of fetalhemoglobin expression relative to cells differentiated from unmodifiedcells. In embodiments, the increase in level of fetal hemoglobin is atleast about 20%, at least about 30%, at least about 40% or at leastabout 50%. Alternatively, or in addition, a cell of the invention maygive rise, e.g., differentiate into, another type of cell, e.g., anerythrocyte, that has a reduced level of beta globin (e.g., hemoglobinbeta comprising a sickling mutation, also referred to herein as sicklebeta globin) expression relative to cells differentiated from unmodifiedcells. In embodiments, the decrease in level of sickle beta-globin is atleast about 20%, at least about 30%, at least about 40% or at leastabout 50%.

The cells of the invention include cells in which expression of one ormore genes has been altered, for example, reduced or inhibited, byintroduction of a CRISPR system comprising a gRNA of the invention. Forexample, the cells of the present invention may have a reduced level ofhemoglobin beta, for example a mutated or wild-type hemoglobin beta,expression relative to unmodified cells. In another aspect, theinvention provides cells which are derived from, e.g., differentiatedfrom, cells in which a CRISPR system comprising a gRNA of the inventionhas been introduced. In such aspects, the cells in which the CRISPRsystem comprising the gRNA of the invention has been introduced may notexhibit the reduced level of hemoglobin beta, for example a mutated orwild-type hemoglobin beta, but the cells derived from, e.g.,differentiated from, said cells exhibit the reduced level of hemoglobinbeta, for example a mutated or wild-type hemoglobin beta. Inembodiments, the derivation, e.g., differentiation, is accomplished invivo (e.g., in a patient, e.g., in a hemoglobinopathy patient, e.g., ina patient with sickle cell disease or a thalassemia, e.g., betathalassemia). In embodiments the cells in which the CRISPR systemcomprising the gRNA of the invention has been introduced are CD34+ cellsand the cells derived, e.g., differentiated, therefrom are of theerythroid lineage, e.g., red blood cells.

The cells of the invention include cells in which expression of one ormore genes has been altered, for example, increased or promoted, byintroduction of a CRISPR system comprising a gRNA of the invention. Forexample, the cells of the present invention may have an increased levelof fetal hemoglobin expression relative to unmodified cells. In anotheraspect, the invention provides cells which are derived from, e.g.,differentiated from, cells in which a CRISPR system comprising a gRNA ofthe invention has been introduced. In such aspects, the cells in whichthe CRISPR system comprising the gRNA of the invention has beenintroduced may not exhibit the increased level of fetal hemoglobin butthe cells derived from, e.g., differentiated from, said cells exhibitthe increased level of fetal hemoglobin. In embodiments, the derivation,e.g., differentiation, is accomplished in vivo (e.g., in a patient,e.g., in a hemoglobinopathy patient, e.g., in a patient with sickle celldisease or a thalassemia, e.g., beta thalassemia). In embodiments thecells in which the CRISPR system comprising the gRNA of the inventionhas been introduced are CD34+ cells and the cells derived, e.g.,differentiated, therefrom are of the erythroid lineage, e.g., red bloodcells.

In another aspect, the invention relates to cells which include an indelat (e.g., within) or near (e.g., within 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides of) a nucleic acidsequence having complementarity to the gRNA molecule (e.g., the targetsequence of the gRNA molecule) or gRNA molecules introduced into saidcells. In embodiments, the indel is a frameshift indel. In embodiments,the cell includes a large deletion, for example a deletion of 1 kb, 2kb, 3 kb, 4 kb, 5 kb, 6 kb or more. In embodiments, the large deletioncomprises nucleic acids disposed between two binding sites for the gRNAmolecule or gRNA molecules introduced into said cells. In embodiments,the deletion comprises, e.g., consists of, the about 4900 nt disposedbetween the target sequence of a gRNA described herein disposed in theHBG1 promoter region and the target sequence of a gRNA described hereindisposed in the HBG2 promoter region. In embodiments, the indel, e.g.,deletion, does not comprise a nucleotide disposed between 5,250,092 and5,249,833, − strand (hg38).

In an aspect, the invention relates to a population of cells (e.g., asdescribed herein), e.g., a population of HSPCs, which comprises cellswhich include an indel at or near (e.g., within 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides of) anucleic acid sequence having complementarity to a gRNA molecule or gRNAmolecules, e.g., as described herein, introduced into said cells, e.g.,as described herein. In embodiments, the indel is a frameshift indel. Inembodiments, the cell population includes cells which comprise a largedeletion, for example a deletion of 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kbor more. In embodiments, the large deletion comprises nucleic acidsdisposed between two binding sites for the gRNA molecule or gRNAmolecules introduced into said cells. In embodiments, the deletioncomprises, e.g., consists of, the about 4900 nt disposed between thetarget sequence of a gRNA described herein disposed in the HBG1 promoterregion and the target sequence of a gRNA described herein disposed inthe HBG2 promoter region. In embodiments, less than 1%, 0.5%, 0.1% or0.001% of the cells of the population (e.g., no cell of the population)comprises a deletion of a nucleotide disposed between 5,250,092 and5,249,833, − strand (hg38). In embodiments, 20%-100% of the cells of thepopulation include said large deletion, indel or indels. In embodiments,30%-100% of the cells of the population include said large deletion,indel or indels. In embodiments, 40%-100% of the cells of the populationinclude said large deletion, indel or indels. In embodiments, 50%-100%of the cells of the population include said large deletion, indel orindels. In embodiments, 60%-100% of the cells of the population includesaid large deletion, indel or indels. In embodiments, 70%-100% of thecells of the population include said large deletion, indel or indels. Inembodiments, 80%-100% of the cells of the population include said largedeletion, indel or indels. In embodiments, 90%-100% of the cells of thepopulation include said large deletion, indel or indels. In embodiments,the population of cells retains the ability to differentiate intomultiple cell types, e.g., maintains the ability to differentiate intocells of erythroid lineage, e.g., red blood cells, e.g., in a subject,e.g., a human. In embodiments, the edited cells (e.g., HSPC cells, e.g.,CD34+ cell, e.g., any subpopulation of CD34+ cell, e.g., as describedherein) maintain the ability (and/or do) to proliferate, e.g., in cellculture, e.g., proliferate at least 5-fold, at least 10-fold, at least15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more,e.g., after 1, 2, 3, 4, 5, 6, 7 or more days (e.g., after about 1 orabout 2 days) in cell culture, e.g., in a cell culture medium describedherein, e.g., a cell culture medium comprising one or more stem cellexpanders, e.g., compound 4. In embodiments, the edited anddifferentiated cells (e.g., red blood cells) maintain the ability toproliferate, e.g., proliferate at least 5-fold, at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, atleast 35-fold, at least 40-fold, at least 45-fold, at least 50-fold ormore after 7 days in erythroid differentiation medium (EDM), e.g., asdescribed in the Examples, and/or, proliferate at least 30-fold, atleast 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, atleast 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, atleast 75-fold, at least 80-fold, at least 85-fold, at least 90-fold, atleast 95-fold, at least 100-fold, at least 110-fold, at least 120-fold,at least 130-fold, at least 140-fold, at least 150-fold, at least200-fold, at least 300-fold, at least 400-fold, at least 500-fold, atleast 600-fold, at least 700-fold, at least 800-fold, at least 900-fold,at least 1000-fold, at least 1100-fold, at least 1200-fold, at least1300-fold, at least 1400-fold, at least 1500-fold or more after 21 days,e.g., in erythroid differentiation medium (EDM), e.g., as described inthe Examples or in a subject (e.g., a mammal, e.g., a human)

In an embodiment, the invention provides a population of cells, e.g.,CD34+ cells, of which at least 90%, e.g., at least 95%, e.g., at least98%, of the cells of the population comprise a large deletion or one ormore indels, e.g., as described herein. Without being bound by theory,it is believed that introduction of a gRNA molecule or CRISPR system asdescribed herein into a population of cells produces a pattern of indelsand/or large deletions in said population, and thus, each cell of thepopulation which comprises an indel and/or large deletion may notexhibit the same indel and/or large deletion. In embodiments, the indeland/or large deletion comprises one or more nucleic acids at or near asite complementary to the targeting domain of a gRNA molecule describedherein; wherein said cells maintain the ability to differentiate intocells of an erythroid lineage, e.g., red blood cells; and/or whereinsaid cells differentiated from the population of cells have an increasedlevel of fetal hemoglobin (e.g., the population has a higher % F cells)relative to cells differentiated from a similar population of unmodifiedcells. In embodiments, the population of cells has undergone at least a2-fold expansion ex vivo, e.g., in the media comprising one or more stemcell expanders, e.g., comprising(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol.In embodiments, the population of cells has undergone at least a 5-foldexpansion ex vivo, e.g., in the media comprising one or more stem cellexpanders, e.g., comprising(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol.

In embodiments, the indel is less than about 50 nucleotides, e.g., lessthan about 45, less than about 40, less than about 35, less than about30 or less than about 25 nucleotides. In embodiments, the indel is lessthan about 25 nucleotides. In embodiments, the indel is less than about20 nucleotides. In embodiments, the indel is less than about 15nucleotides. In embodiments, the indel is less than about 10nucleotides. In embodiments, the indel is less than about 9 nucleotides.In embodiments, the indel is less than about 9 nucleotides. Inembodiments, the indel is less than about 7 nucleotides. In embodiments,the indel is less than about 6 nucleotides. In embodiments, the indel isless than about 5 nucleotides. In embodiments, the indel is less thanabout 4 nucleotides. In embodiments, the indel is less than about 3nucleotides. In embodiments, the indel is less than about 2 nucleotides.In any of the aforementioned embodiments, the indel is at least 1nucleotide. In embodiments, the indel is 1 nucleotide. In embodiments,the large deletion comprises about 1 kb of DNA. In embodiments, thelarge deletion comprises about 2 kb of DNA. In embodiments, the largedeletion comprises about 3 kb of DNA. In embodiments, the large deletioncomprises about 4 kb of DNA. In embodiments, the large deletioncomprises about 5 kb of DNA. In embodiments, the large deletioncomprises about 6 kb of DNA. In embodiments, the large deletioncomprises about 4.9 kb of DNA, for example, disposed between a targetsequence in the HBG1 promoter region and a target sequence in the HBG2promoter region.

In embodiments, a population of cells (e.g., as described herein)comprises a pattern of indels and/or large deletions comprising any 1,2, 3, 4, 5, or 6 of the most frequently detected indels associated witha CRISPR system comprising a gRNA molecule described herein, e.g.,comprises 1, 2, 3, 4, 5, or 6 of the indels and large deletionsdescribed in Table 7-2 (e.g., comprises 1, 2, 3, 4, 5 or 6 of the indelsand large deletions detected at or near the HBG1 target sequence and/orcomprises 1, 2, 3, 4, 5 or 6 of the indels and large deletions detectedat or near the HBG2 target sequence). In embodiments, the indels and/orlarge deletions are detected by a method described herein, e.g., by NGSor qPCR.

In an aspect, the cell or population of cells (e.g., as describedherein) does not comprise an indel or large deletion at an off-targetsite, e.g., as detected by a method described herein.

In embodiments, the progeny, e.g., differentiated progeny, e.g.,erythroid (e.g., red blood cell) progeny of the cell or population ofcells described herein (e.g., derived from a sickle cell diseasepatient) produce a lower level of sickle beta globin and/or a higherlevel of gamma globin than unmodified cells. In embodiments, theprogeny, e.g., differentiated progeny, e.g., erythroid (e.g., red bloodcell) progeny of the cell or population of cells described herein (e.g.,derived from a sickle cell disease patient) produce a lower level ofsickle beta globin and a higher level of gamma globin than unmodifiedcells. In embodiments, sickle beta globin is produced at a level atleast about 20%, at least about 30%, at least about 40% or at leastabout 50% lower than unmodified cells. In embodiments, gamma globin isproduced at a level at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60% or at least about 70%higher than unmodified cells.

In an aspect, the invention provides a population of modified HSPCs orerythroid cells differentiated from said HSPCs (e.g., differentiated exvivo or in a patient), e.g., as described herein, wherein at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95% of the cells are F cells. In embodiments, the population ofcells contains (or is capable of differentiating, e.g., in vivo, into apopulation of erythrocytes that contains) a higher percent of F cellsthan a similar population of cells which have not had a gRNA molecule orgRNA molecules, e.g., as described herein, introduced into said cells.In embodiments, the population of cells has (or is capable ofdifferentiating, e.g., in vivo, into a population of erythrocytes thathas) at least a 20% increase, e.g, at least 21% increase, at least 22%increase, at least 23% increase, at least 24% increase, at least 25%increase, at least 26% increase, at least 27% increase, at least 28%increase, or at least 29% increase, in F cells relative to the similarpopulation of cells which have not had a gRNA molecule or gRNAmolecules, e.g., as described herein, introduced into said cells. Inembodiments, the population of cells has (or is capable ofdifferentiating, e.g., in vivo, into a population of erythrocytes thathas) at least a 30% increase, e.g., at least a 35% increase, at least a40% increase, at least a 45% increase, at least a 50% increase, at leasta 55% increase, at least a 60% increase, at least a 65% increase, atleast a 70% increase, at least a 75% increase, at least a 80% increase,at least a 85% increase, at least a 90% increase or at least a 95%increase, in F cells relative to the similar population of cells whichhave not had a gRNA molecule or gRNA molecules, e.g., as describedherein, introduced into said cells. In embodiments, the population ofcells has (or is capable of differentiating, e.g., in vivo, into apopulation of erythrocytes that has) at a 10-90%, a 20%-80%, a 20%-70%,a 20%-60%, a 20%-50%, a 20%-40%, a 20%-30%, a 25%-80%, a 25%-70%, a25%-60%, a 25%-50%, a 25%-40%, a 25%-35%, a 25%-30%, a 30%-80%, a30%-70%, a 30%-60%, a 30%-50%, a 30%-40%, or a 30%-35% increase in Fcells relative to the similar population of cells which have not had agRNA molecule or gRNA molecules, e.g., as described herein, introducedinto said cells. In embodiments, the population of cells, e.g., asproduced by a method described herein, comprises a sufficient number orcells and/or a sufficient increase in % F cells to treat ahemoglobinopathy, e.g., as described herein, e.g., sickle cell diseaseand/or beta thalassemia, in a patient in need thereof when introducedinto said patient, e.g., in a therapeutically effective amount. Inembodiments, the increase in F cells is as measured in an erythroiddifferentiation assay, e.g., as described herein.

In embodiments, including in any of the embodiments and aspectsdescribed herein, the invention relates to a cell, e.g., a population ofcells, e.g., as modified by any of the gRNA, methods and/or CRISPRsystems described herein, comprising F cells that produce at least 6picograms fetal hemoglobin per cell. In embodiments, the F cells produceat least 7 picograms fetal hemoglobin per cell. In embodiments, the Fcells produce at least 8 picograms fetal hemoglobin per cell. Inembodiments, the F cells produce at least 9 picograms fetal hemoglobinper cell. In embodiments, the F cells produce at least 10 picogramsfetal hemoglobin per cell. In embodiments, the F cells produce anaverage of between 6.0 and 7.0 picograms, between 7.0 and 8.0, between8.0 and 9.0, between 9.0 and 10.0, between 10.0 and 11.0, or between11.0 and 12.0 picograms of fetal hemoglobin per cell.

In embodiments, a cell or population of cells, e.g., as described herein(for example, comprising an indel, e.g., a large deletion or indeldescribed in Table 7-2) (or its progeny), is detectable in the cells ofa subject to which it is introduced, for example, remains detectible bydetecting the indel, for example, using a method described herein. Inembodiments, the cell or population of cells (or its progeny) isdetectible in a subject to which it is introduced for at least 10 weeks,at least 14 weeks, at least 16 weeks, at least 18 weeks, at least 20weeks, at least 30 weeks at least 40 weeks, at least 50 weeks, or longerafter said cell or population of cells is introduced into said subject.

In embodiments, one or more indels (e.g., a large deletion or indeldescribed in Table 7-2), is detectable in the cells (e.g., the cells,e.g., CD34+ cells, of the bone marrow and/or peripheral blood) of asubject to which the cells or population of cells described herein havebeen introduced, for example, remains detectible by a method describedherein, e.g., NGS. In embodiments, the one or more indels is detectiblein the cells (e.g., the cells, e.g., CD34+ cells, of the bone marrowand/or peripheral blood) of a subject to which the cells or populationof cells described herein have been introduced for at least 10 weeks, atleast 14 weeks, at least 16 weeks, at least 18 weeks, at least 20 weeks,at least 30 weeks at least 40 weeks, at least 50 weeks, or longer afterthe cell or population of cells described herein is introduced into saidsubject. In embodiments, the level of detection of said one or moreindels does not decrease over time, or decreases by less than 5%, lessthan 10%, less than 15%, less than 20%, less than 30%, less than 40% orless than 50% (for example relative to the level of indel detectionpre-transplant or relative to the level of detection at week 2post-transplant or at week 8 post transplant), for example when measuredat week 20 post-transplant relative to the level of detection (e.g.,percentage of cells comprising the one or more indels) measuredpre-transplant or measured at week 2 post transplant or at week 8 posttransplant.

In embodiments, including in any of the aforementioned embodiments, thecell and/or population of cells of the invention includes, e.g.,consists of, cells which do not comprise nucleic acid encoding a Cas9molecule.

Methods of Treatment

Delivery Timing

In an embodiment, one or more nucleic acid molecules (e.g., DNAmolecules) other than the components of a Cas system, e.g., the Cas9molecule component and/or the gRNA molecule component described herein,are delivered. In an embodiment, the nucleic acid molecule is deliveredat the same time as one or more of the components of the Cas system aredelivered. In an embodiment, the nucleic acid molecule is deliveredbefore or after (e.g., less than about 30 minutes, 1 hour, 2 hours, 3hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2weeks, or 4 weeks) one or more of the components of the Cas system aredelivered. In an embodiment, the nucleic acid molecule is delivered by adifferent means than one or more of the components of the Cas system,e.g., the Cas9 molecule component and/or the gRNA molecule component,are delivered. The nucleic acid molecule can be delivered by any of thedelivery methods described herein. For example, the nucleic acidmolecule can be delivered by a viral vector, e.g., anintegration-deficient lentivirus, and the Cas9 molecule component and/orthe gRNA molecule component can be delivered by electroporation, e.g.,such that the toxicity caused by nucleic acids (e.g., DNAs) can bereduced. In an embodiment, the nucleic acid molecule encodes atherapeutic protein, e.g., a protein described herein. In an embodiment,the nucleic acid molecule encodes an RNA molecule, e.g, an RNA moleculedescribed herein.

Bi-Modal or Differential Delivery of Components

Separate delivery of the components of a Cas system, e.g., the Cas9molecule component and the gRNA molecule component, and moreparticularly, delivery of the components by differing modes, can enhanceperformance, e.g., by improving tissue specificity and safety. In anembodiment, the Cas9 molecule and the gRNA molecule are delivered bydifferent modes, or as sometimes referred to herein as differentialmodes. Different or differential modes, as used herein, refer modes ofdelivery, that confer different pharmacodynamic or pharmacokineticproperties on the subject component molecule, e.g., a Cas9 molecule,gRNA molecule, template nucleic acid, or payload. E.g., the modes ofdelivery can result in different tissue distribution, differenthalf-life, or different temporal distribution, e.g., in a selectedcompartment, tissue, or organ.

Some modes of delivery, e.g., delivery by a nucleic acid vector thatpersists in a cell, or in progeny of a cell, e.g., by autonomousreplication or insertion into cellular nucleic acid, result in morepersistent expression of and presence of a component. Examples includeviral, e.g., adeno associated virus or lentivirus, delivery.

By way of example, the components, e.g., a Cas9 molecule and a gRNAmolecule, can be delivered by modes that differ in terms of resultinghalf life or persistent of the delivered component the body, or in aparticular compartment, tissue or organ. In an embodiment, a gRNAmolecule can be delivered by such modes. The Cas9 molecule component canbe delivered by a mode which results in less persistence or lessexposure of its to the body or a particular compartment or tissue ororgan.

More generally, in an embodiment, a first mode of delivery is used todeliver a first component and a second mode of delivery is used todeliver a second component. The first mode of delivery confers a firstpharmacodynamic or pharmacokinetic property. The first pharmacodynamicproperty can be, e.g., distribution, persistence, or exposure, of thecomponent, or of a nucleic acid that encodes the component, in the body,a compartment, tissue or organ. The second mode of delivery confers asecond pharmacodynamic or pharmacokinetic property. The secondpharmacodynamic property can be, e.g., distribution, persistence, orexposure, of the component, or of a nucleic acid that encodes thecomponent, in the body, a compartment, tissue or organ.

In an embodiment, the first pharmacodynamic or pharmacokinetic property,e.g., distribution, persistence or exposure, is more limited than thesecond pharmacodynamic or pharmacokinetic property.

In an embodiment, the first mode of delivery is selected to optimize,e.g., minimize, a pharmacodynamic or pharmacokinetic property, e.g.,distribution, persistence or exposure.

In an embodiment, the second mode of delivery is selected to optimize,e.g., maximize, a pharmacodynamic or pharmcokinetic property, e.g.,distribution, persistence or exposure.

In an embodiment, the first mode of delivery comprises the use of arelatively persistent element, e.g., a nucleic acid, e.g., a plasmid orviral vector, e.g., an AAV or lentivirus. As such vectors are relativelypersistent product transcribed from them would be relatively persistent.

In an embodiment, the second mode of delivery comprises a relativelytransient element, e.g., an RNA or protein.

In an embodiment, the first component comprises gRNA, and the deliverymode is relatively persistent, e.g., the gRNA is transcribed from aplasmid or viral vector, e.g., an AAV or lentivirus. Transcription ofthese genes would be of little physiological consequence because thegenes do not encode for a protein product, and the gR As are incapableof acting in isolation. The second component, a Cas9 molecule, isdelivered in a transient manner, for example as mRNA or as protein,ensuring that the full Cas9 molecule/gRNA molecule complex is onlypresent and active for a short period of time.

Furthermore, the components can be delivered in different molecular formor with different delivery vectors that complement one another toenhance safety and tissue specificity.

Use of differential delivery modes can enhance performance, safety andefficacy. For example, the likelihood of an eventual off-targetmodification can be reduced. Delivery of immunogenic components, e.g.,Cas9 molecules, by less persistent modes can reduce immunogenicity, aspeptides from the bacterially-derived Cas enzyme are displayed on thesurface of the cell by MHC molecules. A two-part delivery system canalleviate these drawbacks.

Differential delivery modes can be used to deliver components todifferent, but overlapping target regions. The formation active complexis minimized outside the overlap of the target regions. Thus, in anembodiment, a first component, e.g., a gRNA molecule is delivered by afirst delivery mode that results in a first spatial, e.g., tissue,distribution. A second component, e.g., a Cas9 molecule is delivered bya second delivery mode that results in a second spatial, e.g., tissue,distribution. In an embodiment, the first mode comprises a first elementselected from a liposome, nanoparticle, e.g., polymeric nanoparticle,and a nucleic acid, e.g., viral vector. The second mode comprises asecond element selected from the group. In an embodiment, the first modeof delivery comprises a first targeting element, e.g., a cell specificreceptor or an antibody, and the second mode of delivery does notinclude that element. In an embodiment, the second mode of deliverycomprises a second targeting element, e.g., a second cell specificreceptor or second antibody.

When the Cas9 molecule is delivered in a virus delivery vector, aliposome, or polymeric nanoparticle, there is the potential for deliveryto and therapeutic activity in multiple tissues, when it may bedesirable to only target a single tissue. A two-part delivery system canresolve this challenge and enhance tissue specificity. If the gRNAmolecule and the Cas9 molecule are packaged in separated deliveryvehicles with distinct but overlapping tissue tropism, the fullyfunctional complex is only be formed in the tissue that is targeted byboth vectors.

Candidate Cas molecules, e.g., Cas9 molecules, candidate gRNA molecules,candidate Cas9 molecule/gRNA molecule complexes, and candidate CRISPRsystems, can be evaluated by art-known methods or as described herein.For example, exemplary methods for evaluating the endonuclease activityof Cas9 molecule are described, e.g., in Jinek el al., SCIENCE 2012;337(6096):816-821.

Additional aspects are described in the enumerated embodiments, below.

EMBODIMENTS

1. A gRNA molecule comprising a tracr and crRNA, wherein the crRNAcomprises a targeting domain that:

a) is complementary with a target sequence of a nondeletional HFPHregion (e.g., a human nondeletional HPFH region);

b) is complementary with a target sequence within the genomic nucleicacid sequence at Chr11:5,249,833 to Chr11:5,250,237, − strand, hg38;

c) is complementary with a target sequence within the genomic nucleicacid sequence at Chr11:5,254,738 to Chr11:5,255,164, − strand, hg38;

d) is complementary with a target sequence within the genomic nucleicacid sequence at Chr11:5,250,094-5,250,237, − strand, hg38;

e) is complementary with a target sequence within the genomic nucleicacid sequence at Chr11:5,255,022-5,255,164, − strand, hg38;

f) is complementary with a target sequence within the genomic nucleicacid sequence at Chr11:5,249,833-5,249,927, − strand, hg38;

g) is complementary with a target sequence within the genomic nucleicacid sequence at Chr11:5,254,738-5,254,851, − strand, hg38;

h) is complementary with a target sequence within the genomic nucleicacid sequence at Chr11:5,250,139-5,250,237, − strand, hg38; or

i) combinations thereof.

2. A gRNA molecule of embodiment 1, wherein the targeting domaincomprises, e.g., consists of, any one of SEQ ID NO: 1 to SEQ ID NO: 72,or a fragment thereof.

3. A gRNA molecule of embodiment 2, wherein the targeting domaincomprises, e.g., consists of, any one of SEQ ID NO: 1, SEQ ID NO: 6, SEQID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 28, SEQ ID NO: 34, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47,SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO:54, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 67, or afragment thereof.

4. A gRNA molecule of embodiment 2, wherein the targeting domaincomprises, e.g., consists of, any one of

a) SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO:48, SEQ ID NO: 51, SEQ ID NO: 67, or a fragment thereof; or

b) SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 54, or a fragment thereof.

5. The gRNA molecule of any of embodiments 2-4, wherein the targetingdomain comprises, e.g., consists of, 17, 18, 19, or 20 consecutivenucleic acids of any one of the recited targeting domain sequences.

6. The gRNA molecule of embodiment 5, wherein the 17, 18, 19, or 20consecutive nucleic acids of any one of the recited targeting domainsequences are the 17, 18, 19, or 20 consecutive nucleic acids disposedat the 3′ end of the recited targeting domain sequence.

7. The gRNA molecule of embodiment 5, wherein the 17, 18, 19, or 20consecutive nucleic acids of any one of the recited targeting domainsequences are the 17, 18, 19, or 20 consecutive nucleic acids disposedat the 5′ end of the recited targeting domain sequence.

8. The gRNA molecule of embodiment 5, wherein the 17, 18, 19, or 20consecutive nucleic acids of any one of the recited targeting domainsequences do not comprise either the 5′ or 3′ nucleic acid of therecited targeting domain sequence.

9. The gRNA molecule of any of embodiments 2-8, wherein the targetingdomain consists of the recited targeting domain sequence.

10. The gRNA molecule of any of the previous embodiments, wherein aportion of the crRNA and a portion of the tracr hybridize to form aflagpole comprising SEQ ID NO: 182 or 183.

11. The gRNA molecule of embodiment 10, wherein the flagpole furthercomprises a first flagpole extension, located 3′ to the crRNA portion ofthe flagpole, wherein said first flagpole extension comprises SEQ ID NO:184.

12. The gRNA molecule of embodiment 10 or 11, wherein the flagpolefurther comprises a second flagpole extension located 3′ to the crRNAportion of the flagpole and, if present, the first flagpole extension,wherein said second flagpole extension comprises SEQ ID NO: 185.

13. The gRNA molecule of any of embodiments 1-12, wherein the tracrcomprises SEQ ID NO: 224 or SEQ ID NO: 225.

14. The gRNA molecule of any of embodiments 1-13, wherein the tracrcomprises SEQ ID NO: 232, optionally further comprising, at the 3′ end,an additional 1, 2, 3, 4, 5, 6, or 7 uracil (U) nucleotides.

15. The gRNA molecule of any of embodiments 1-14, wherein the crRNAcomprises, from 5′ to 3′, [targeting domain]−:

a) SEQ ID NO: 182;

b) SEQ ID NO: 183;

c) SEQ ID NO: 199;

d) SEQ ID NO: 200;

e) SEQ ID NO: 201;

f) SEQ ID NO: 202; or

g) SEQ ID NO: 226.

16. The gRNA molecule of any of embodiments 1-9 or 15, wherein the tracrcomprises, from 5′ to 3′:

a) SEQ ID NO: 187;

b) SEQ ID NO: 188;

c) SEQ ID NO: 203;

d) SEQ ID NO: 204;

e) SEQ ID NO: 224;

f) SEQ ID NO: 225;

g) SEQ ID NO: 232;

h) SEQ ID NO: 227;

i) (SEQ ID NO: 228;

j) SEQ ID NO: 229;

k) any of a) to j), above, further comprising, at the 3′ end, at least1, 2, 3, 4, 5, 6 or 7 uracil (U) nucleotides, e.g., 1, 2, 3, 4, 5, 6, or7 uracil (U) nucleotides;

l) any of a) to k), above, further comprising, at the 3′ end, at least1, 2, 3, 4, 5, 6 or 7 adenine (A) nucleotides, e.g., 1, 2, 3, 4, 5, 6,or 7 adenine (A) nucleotides; or

m) any of a) to l), above, further comprising, at the 5′ end (e.g., atthe 5′ terminus), at least 1, 2, 3, 4, 5, 6 or 7 adenine (A)nucleotides, e.g., 1, 2, 3, 4, 5, 6, or 7 adenine (A) nucleotides.

17. The gRNA molecule of any of embodiments 1-9, wherein the targetingdomain and the tracr are disposed on separate nucleic acid molecules,and wherein the nucleic acid molecule comprising the targeting domaincomprises SEQ ID NO: 201, optionally disposed immediately 3′ to thetargeting domain, and the nucleic acid molecule comprising the tracrcomprises, e.g., consists of, SEQ ID NO: 224.

18. The gRNA molecule of any of embodiments 13-14, wherein the crRNAportion of the flagpole comprises SEQ ID NO: 201 or SEQ ID NO: 202.

19. The gRNA molecule of any of embodiments 1-12, wherein the tracrcomprises SEQ ID NO: 187 or 188, and optionally, if a first flagpoleextension is present, a first tracr extension, disposed 5′ to SEQ ID NO:187 or 188, said first tracr extension comprising SEQ ID NO: 189.

20. The gRNA molecule of any of embodiments 1-19, wherein the targetingdomain and the tracr are disposed on separate nucleic acid molecules.

21. The gRNA molecule of any of embodiments 1-19, wherein the targetingdomain and the tracr are disposed on a single nucleic acid molecule, andwherein the tracr is disposed 3′ to the targeting domain.

22. The gRNA molecule of embodiment 21, further comprising a loop,disposed 3′ to the targeting domain and 5′ to the tracr.

23. The gRNA molecule of embodiment 22, wherein the loop comprises SEQID NO: 186.

24. The gRNA molecule of any of embodiments 1-9, comprising, from 5′ to3′, [targeting domain]−:

(a) SEQ ID NO: 195;

(b) SEQ ID NO: 196;

(c) SEQ ID NO: 197;

(d) SEQ ID NO: 198;

(e) SEQ ID NO: 231; or

(f) any of (a) to (e), above, further comprising, at the 3′ end, 1, 2,3, 4, 5, 6 or 7 uracil (U) nucleotides.

25. The gRNA molecule of any of embodiments 1-9 or 21-24, wherein thetargeting domain and the tracr are disposed on a single nucleic acidmolecule, and wherein said nucleic acid molecule comprises, e.g.,consists of, said targeting domain and SEQ ID NO: 231, optionallydisposed immediately 3′ to said targeting domain.

26. The gRNA molecule of any of embodiments 1-25 wherein one, oroptionally more than one, of the nucleic acid molecules comprising thegRNA molecule comprises:

a) one or more, e.g., three, phosphorothioate modifications at the 3′end of said nucleic acid molecule or molecules;

b) one or more, e.g., three, phosphorothioate modifications at the 5′end of said nucleic acid molecule or molecules;

c) one or more, e.g., three, 2′-O-methyl modifications at the 3′ end ofsaid nucleic acid molecule or molecules;

d) one or more, e.g., three, 2′-O-methyl modifications at the 5′ end ofsaid nucleic acid molecule or molecules;

e) a 2′ O-methyl modification at each of the 4^(th)-to-terminal,3^(rd)-to-terminal, and 2^(nd)-to-terminal 3′ residues of said nucleicacid molecule or molecules;

f) a 2′ O-methyl modification at each of the 4^(th)-to-terminal,3^(rd)-to-terminal, and 2^(nd)-to-terminal 5′ residues of said nucleicacid molecule or molecules; or

f) any combination thereof.

27. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 74;

(b) SEQ ID NO: 75; or

(c) SEQ ID NO: 76.

28. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 77, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 77, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 78, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 78, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

29. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 79;

(b) SEQ ID NO: 80; or

(c) SEQ ID NO: 81.

30. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 82, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 82, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 83, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 83, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

31. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 84;

(b) SEQ ID NO: 85; or

(c) SEQ ID NO: 86.

32. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 87, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 87, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 88, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 88, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

33. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 89;

(b) SEQ ID NO: 90; or

(c) SEQ ID NO: 91.

34. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 92, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 92, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 93, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 93, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

35. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 94;

(b) SEQ ID NO: 95; or

(c) SEQ ID NO: 96.

36. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 97, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 97, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 98, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 98, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

37. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 99;

(b) SEQ ID NO: 100; or

(c) SEQ ID NO: 101.

38. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 102, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 102, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 103, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 103, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

39. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 104;

(b) SEQ ID NO: 105; or

(c) SEQ ID NO:106.

40. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 107, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 107, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 108, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 108, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

41. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 109;

(b) SEQ ID NO: 110; or

(c) SEQ ID NO: 111.

42. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 112, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 112, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 113, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 113, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

43. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 114;

(b) SEQ ID NO: 115; or

(c) SEQ ID NO:116.

44. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 117, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 117, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 118, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 118, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

45. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 119;

(b) SEQ ID NO: 120; or

(c) SEQ ID NO: 121.

46. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 122, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 122, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 123, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 123, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

47. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 124;

(b) SEQ ID NO: 125; or

(c) SEQ ID NO:126.

48. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 127, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 127, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 128, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 128, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

49. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 129;

(b) SEQ ID NO: 130; or

(c) SEQ ID NO: 131.

50. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 132, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 132, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 133, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 133, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

51. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 134;

(b) SEQ ID NO: 135; or

(c) SEQ ID NO:136.

52. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 137, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 137, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 138, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 138, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

53. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 139;

(b) SEQ ID NO: 140; or

(c) SEQ ID NO: 141.

54. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 142, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 142, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 143, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 143, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

55. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 144;

(b) SEQ ID NO: 145; or

(c) SEQ ID NO:146.

56. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 147, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 147, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 148, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 148, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

57. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 149;

(b) SEQ ID NO: 150; or

(c) SEQ ID NO: 151.

58. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 152, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 152, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 153, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 153, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

59. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 154;

(b) SEQ ID NO: 155; or

(c) SEQ ID NO:156.

60. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 157, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 157, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 158, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 158, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

61. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 159;

(b) SEQ ID NO: 160; or

(c) SEQ ID NO: 161.

62. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 162, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 162, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 163, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 163, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

63. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 164;

(b) SEQ ID NO: 165; or

(c) SEQ ID NO:166.

64. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 167, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 167, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 168, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 168, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

65. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 169;

(b) SEQ ID NO: 170; or

(c) SEQ ID NO: 171.

66. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 172, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 172, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 173, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 173, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

67. A gRNA molecule of embodiment 1, comprising, e.g., consisting of,the sequence:

(a) SEQ ID NO: 174;

(b) SEQ ID NO: 175; or

(c) SEQ ID NO:176.

68. A gRNA molecule of embodiment 1, comprising, e.g., consisting of:

(a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 177, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224;

(b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 177, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73;

(c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 178, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 224; or

(d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 178, and a tracrcomprising, e.g., consisting of, SEQ ID NO: 73.

69. A gRNA molecule of any of embodiments 1-68, wherein

a) when a CRISPR system (e.g., an RNP as described herein) comprisingthe gRNA molecule is introduced into a cell, an indel is formed at ornear the target sequence complementary to the targeting domain of thegRNA molecule; and/or

b) when a CRISPR system (e.g., an RNP as described herein) comprisingthe gRNA molecule is introduced into a cell, a deletion is createdcomprising sequence, e.g., comprising substantially all the sequence,between a sequence complementary to the gRNA targeting domain (e.g., atleast 90% complementary to the gRNA targeting domain, e.g., fullycomplementary to the gRNA targeting domain) in the HBG1 promoter regionand a sequence complementary to the gRNA targeting domain (e.g., atleast 90% complementary to the gRNA targeting domain, e.g., fullycomplementary to the gRNA targeting domain) in the HBG2 promoter region.

70. The gRNA molecule of embodiment 69, wherein the indel does notcomprise a nucleotide disposed between 5,250,092 and 5,249,833, − strand(hg38), optionally wherein the indel does not comprise a nucleotide of anondeletional HPFH or transcription factor binding site.

71. A gRNA molecule of any of embodiments 1-70, wherein when a CRISPRsystem (e.g., an RNP as described herein) comprising the gRNA moleculeis introduced into a population of cells, an indel is formed at or nearthe target sequence complementary to the targeting domain of the gRNAmolecule in at least about 15%, e.g., at least about 17%, e.g., at leastabout 20%, e.g., at least about 30%, e.g., at least about 40%, e.g., atleast about 50%, e.g., at least about 55%, e.g., at least about 60%,e.g., at least about 70%, e.g., at least about 75%, e.g., at least about80%, e.g., at least about 85%, e.g., at least about 90%, e.g., at leastabout 95%, of the cells of the population.

72. A gRNA molecule of any of embodiments 69-71, wherein the indelcomprises at least one nucleotide of an HBG1 promoter region or at leastone nucleotide of an HBG2 promoter region.

73. A gRNA molecule of any of embodiments 71-72, wherein at least about15% of the cells of the population comprise an indel which comprises atleast one nucleotide of an HBG1 promoter region and an indel whichcomprises at least one nucleotide of an HBG2 promoter region.

74. A gRNA molecule of embodiment 71-73, wherein the percentage of thecells of the population which comprise an indel which comprises at leastone nucleotide of an HBG1 promoter region differs from percentage of thecells of the population which comprise an indel which comprises at leastone nucleotide of an HBG2 promoter region by at least about 5%, e.g., atleast about 10%, e.g., at least about 20%, e.g., at least about 30%.

75. The gRNA molecule of any of embodiments 69-74, wherein the indel isas measured by next generation sequencing (NGS).

76. A gRNA molecule of any of embodiments 1-75, wherein when a CRISPRsystem (e.g., an RNP as described herein) comprising the gRNA moleculeis introduced into a cell, expression of fetal hemoglobin is increasedin said cell or its progeny, e.g., its erythroid progeny, e.g., its redblood cell progeny.

77. A gRNA molecule of embodiment 76, wherein when a CRISPR system(e.g., an RNP as described herein) comprising the gRNA molecule isintroduced into a population of cells, the percentage of F cells in saidpopulation or population of its progeny, e.g., its erythroid progeny,e.g., its red blood cell progeny, is increased by at least about 15%,e.g., at least about 17%, e.g., at least about 20%, e.g., at least about25%, e.g., at least about 30%, e.g., at least about 35%, e.g., at leastabout 40%, relative to the percentage of F cells in a population ofcells to which the gRNA molecule was not introduced or a population ofits progeny, e.g., its erythroid progeny, e.g., its red blood cellprogeny.

78. A gRNA molecule of any of embodiments 76, wherein said cell or itsprogeny, e.g., its erythroid progeny, e.g., its red blood cell progeny,produces at least about 6 picograms (e.g., at least about 7 picograms,at least about 8 picograms, at least about 9 picograms, at least about10 picograms, or from about 8 to about 9 picograms, or from about 9 toabout 10 picograms) fetal hemoglobin per cell.

79. The gRNA molecule of any of embodiments 1-78, wherein when a CRISPRsystem (e.g., an RNP as described herein) comprising the gRNA moleculeis introduced into a cell, no off-target indels are formed in said cell,e.g., no off-target indels are formed outside of the HBG1 and/or HBG2promoter regions, e.g., as detectible by next generation sequencingand/or a nucleotide insertional assay.

80. The gRNA molecule of any of embodiments 1-78, wherein when a CRISPRsystem (e.g., an RNP as described herein) comprising the gRNA moleculeis introduced into a population of cells, no off-target indel, e.g., nooff-target indel outside of the HBG1 and/or HBG2 promoter regions, isdetected in more than about 5%, e.g., more than about 1%, e.g., morethan about 0.1%, e.g., more than about 0.01%, of the cells of thepopulation of cells, e.g., as detectible by next generation sequencingand/or a nucleotide insertional assay.

81. The gRNA molecule of any of embodiments 69-80, wherein the cell is(or population of cells comprises) a mammalian, primate, or human cell,e.g., is a human cell.

82. The gRNA molecule of embodiment 81, wherein the cell is (orpopulation of cells comprises) an HSPC.

83. The gRNA molecule of embodiment 82, wherein the HSPC is CD34+.

84. The gRNA molecule of embodiment 83, wherein the HSPC is CD34+CD90+.

85. The gRNA molecule of any of embodiments 69-84, wherein the cell isautologous with respect to a patient to be administered said cell.

86. The gRNA molecule of any of embodiments 69-84, wherein the cell isallogeneic with respect to a patient to be administered said cell.

87. A composition comprising:

1) one or more gRNA molecules (including a first gRNA molecule) of anyof embodiments 1-86 and a Cas9 molecule;

2) one or more gRNA molecules (including a first gRNA molecule) of anyof embodiments 1-86 and nucleic acid encoding a Cas9 molecule;

3) nucleic acid encoding one or more gRNA molecules (including a firstgRNA molecule) of any of embodiments 1-86 and a Cas9 molecule;

4) nucleic acid encoding one or more gRNA molecules (including a firstgRNA molecule) of any of embodiments 1-86 and nucleic acid encoding aCas9 molecule; or

5) any of 1) to 4), above, and a template nucleic acid; or

6) any of 1) to 4) above, and nucleic acid comprising sequence encodinga template nucleic acid.

88. A composition comprising a first gRNA molecule of any of embodiments1-86, optionally further comprising a Cas9 molecule.

89. The composition of embodiment 87 or 88, wherein the Cas9 molecule isan active or inactive s. pyogenes Cas9.

90. The composition of embodiment 87-89, wherein the Cas9 moleculecomprises SEQ ID NO: 205.

91. The composition of embodiment 87-89, wherein the Cas9 moleculecomprises, e.g., consists of:

(a) SEQ ID NO: 233;

(b) SEQ ID NO: 234;

(c) SEQ ID NO: 235;

(d) SEQ ID NO: 236;

(e) SEQ ID NO: 237;

(f) SEQ ID NO: 238;

(g) SEQ ID NO: 239;

(h) SEQ ID NO: 240;

(i) SEQ ID NO: 241;

(j) SEQ ID NO: 242;

(k) SEQ ID NO: 243; or

(l) SEQ ID NO: 244.

92. The composition of any of embodiments 88-91, wherein the first gRNAmolecule and Cas9 molecule are present in a ribonuclear protein complex(RNP).

93. The composition of any of embodiments 87-92, further comprising asecond gRNA molecule; a second gRNA molecule and a third gRNA molecule;or a second gRNA molecule, optionally, a third gRNA molecule, and,optionally, a fourth gRNA molecule, wherein the second gRNA molecule,the optional third gRNA molecule, and the optional fourth gRNA moleculeare a gRNA molecule of any of embodiments 1-68, and wherein each gRNAmolecule of the composition is complementary to a different targetsequence.

94. The composition of embodiment 93, wherein two or more of the firstgRNA molecule, the second gRNA molecule, the optional third gRNAmolecule, and the optional fourth gRNA molecule are complementary totarget sequences within the same gene or region.

95. The composition of embodiment 93 or 94, wherein the first gRNAmolecule, the second gRNA molecule, the optional third gRNA molecule,and the optional fourth gRNA molecule are complementary to targetsequences not more than 6000 nucleotides, not more than 5000nucleotides, not more than 500, not more than 400 nucleotides, not morethan 300, not more than 200 nucleotides, not more than 100 nucleotides,not more than 90 nucleotides, not more than 80 nucleotides, not morethan 70 nucleotides, not more than 60 nucleotides, not more than 50nucleotides, not more than 40 nucleotides, not more than 30 nucleotides,not more than 20 nucleotides or not more than 10 nucleotides apart.

96. The composition of embodiment 93, wherein two or more of the firstgRNA molecule, the second gRNA molecule, the optional third gRNAmolecule, and the optional fourth gRNA molecule comprise at least onegRNA molecule which comprises a targeting domain complementary to atarget sequence of an HBG1 promoter region and at least one gRNAmolecule which comprises a targeting domain complementary to a targetsequence of an HBG2 promoter region.

97. The composition of any of embodiments 94-95, comprising a first gRNAmolecule and a second gRNA molecule, wherein the first gRNA molecule andsecond gRNA molecule are:

(a) independently selected from the gRNA molecules of embodiment 1, andare complementary to different target sequences;

(b) independently selected from the gRNA molecules of embodiment 2, andare complementary to different target sequences;

c) independently selected from the gRNA molecules of embodiment 3, andare complementary to different target sequences; or

(d) independently selected from the gRNA molecules of embodiment 4, andare complementary to different target sequences; or

(e) independently selected from the gRNA molecules of any of embodiments27-68, and are complementary to different target sequences.

98. The composition of any of embodiments 94-96, comprising a first gRNAmolecule and a second gRNA molecule, wherein:

a) the first gRNA molecule is complementary to a target sequencecomprising at least 1 nucleotide (e.g., comprising 20 consecutivenucleotides) within:

-   -   i) Chr11:5,249,833 to Chr11:5,250,237 (hg38);    -   ii) Chr11:5,250,094-5,250,237 (hg38);    -   iii) Chr11:5,249,833-5,249,927 (hg38); or    -   iv) Chr11:5,250,139-5,250,237 (hg38);

b) the second gRNA molecule is complementary to a target sequencecomprising at least 1 nucleotide (e.g., comprising 20 consecutivenucleotides) within:

-   -   i) Chr11:5,254,738 to Chr11:5,255,164 (hg38);    -   ii) Chr11:5,255,022-5,255,164 (hg38); or    -   iii) Chr11:5,254,738-5,254,851 (hg38).

99. The composition of any of embodiments 87-108, wherein with respectto the gRNA molecule components of the composition, the compositionconsists of a first gRNA molecule and a second gRNA molecule.

100. The composition of any one of embodiments 87-109, wherein each ofsaid gRNA molecules is in a ribonuclear protein complex (RNP) with aCas9 molecule described herein, e.g., a Cas9 molecule of any ofembodiments 90 or 91.

101. The composition of any of embodiments 87-100, comprising a templatenucleic acid, wherein the template nucleic acid comprises a nucleotidethat corresponds to a nucleotide at or near the target sequence of thefirst gRNA molecule.

102. The composition of any of embodiments 101, wherein the templatenucleic acid comprises nucleic acid encoding:

(a) human beta globin, e.g., human beta globin comprising one or more ofthe mutations G16D, E22A and T87Q, or fragment thereof; or

(b) human gamma globin, or fragment thereof.

103. The composition of any of embodiments 87-102, formulated in amedium suitable for electroporation.

104. The composition of any of embodiments 87-103, wherein each of saidgRNA molecules is in a RNP with a Cas9 molecule described herein, andwherein each of said RNP is at a concentration of less than about 10 uM,e.g., less than about 3 uM, e.g., less than about 1 uM, e.g., less thanabout 0.5 uM, e.g., less than about 0.3 uM, e.g., less than about 0.1uM, optionally wherein the concentration of said RNP is about 2 uM or isabout 1 uM, optionally wherein the composition further comprises apopulation of cells, e.g., HSPCs.

105. A nucleic acid sequence that encodes one or more gRNA molecules ofany of embodiments 1-68.

106. The nucleic acid sequence of embodiment 105, wherein the nucleicacid comprises a promoter operably linked to the sequence that encodesthe one or more gRNA molecules.

107. The nucleic acid sequence of embodiment 106, wherein the promoteris a promoter recognized by an RNA polymerase II or RNA polymerase III.

108. The nucleic acid sequence of embodiment 107, wherein the promoteris a U6 promoter or an HI promoter.

109. The nucleic acid sequence of any of embodiments 105-108, whereinthe nucleic acid further encodes a Cas9 molecule.

110. The nucleic acid sequence of embodiment 109, wherein the Cas9molecule comprises any of SEQ ID NO: 205, SEQ ID NO: 233, SEQ ID NO:234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO:243 or SEQ ID NO: 244.

111. The nucleic acid sequence of any of embodiments 109-110, whereinsaid nucleic acid comprises a promoter operably linked to the sequencethat encodes a Cas9 molecule.

112. The nucleic acid sequence of embodiment 111, wherein the promoteris an EF-1 promoter, a CMV IE gene promoter, an EF-1α promoter, anubiquitin C promoter, or a phosphoglycerate kinase (PGK) promoter.

113. A vector comprising the nucleic acid of any of embodiments 105-112.

114. The vector of embodiment 113, wherein in the vector is selectedfrom the group consisting of a lentiviral vector, an adenoviral vector,an adeno-associated viral (AAV) vector, a herpes simplex virus (HSV)vector, a plasmid, a minicircle, a nanoplasmid, and an RNA vector.

115. A method of altering a cell (e.g., a population of cells), (e.g.,altering the structure (e.g., sequence) of nucleic acid) at or near atarget sequence within said cell, comprising contacting (e.g.,introducing into) said cell (e.g., population of cells) with:

1) one or more gRNA molecules of any of embodiments 1-68 and a Cas9molecule;

2) one or more gRNA molecules of any of embodiments 1-68 and nucleicacid encoding a Cas9 molecule;

3) nucleic acid encoding one or more gRNA molecules of any ofembodiments 1-68 and a Cas9 molecule;

4) nucleic acid encoding one or more gRNA molecules of any ofembodiments 1-68 and nucleic acid encoding a Cas9 molecule;

5) any of 1) to 4), above, and a template nucleic acid;

6) any of 1) to 4) above, and nucleic acid comprising sequence encodinga template nucleic acid;

7) the composition of any of embodiments 87-104; or

8) the vector of any of embodiments 113-114.

116. The method of embodiment 115, wherein the gRNA molecule or nucleicacid encoding the gRNA molecule, and the Cas9 molecule or nucleic acidencoding the Cas9 molecule, are formulated in a single composition.

117. The method of embodiment 115, wherein the gRNA molecule or nucleicacid encoding the gRNA molecule, and the Cas9 molecule or nucleic acidencoding the Cas9 molecule, are formulated in more than one composition.

118. The method of embodiment 117, wherein the more than one compositionare delivered simultaneously or sequentially.

119. The method of any of embodiments 115-118, wherein the cell is ananimal cell.

120. The method of any of embodiments 115-118, wherein the cell is amammalian, primate, or human cell.

121. The method of embodiment 120, wherein the cell is a hematopoieticstem or progenitor cell (HSPC) (e.g., a population of HSPCs).

122. The method of any of embodiments 115-121, wherein the cell is aCD34+ cell.

123. The method of any of embodiments 115-122, wherein the cell is aCD34+CD90+ cell.

124. The method of any of embodiments 115-123, wherein the cell isdisposed in a composition comprising a population of cells that has beenenriched for CD34+ cells.

125. The method of any of embodiments 115-124, wherein the cell (e.g.population of cells) has been isolated from bone marrow, mobilizedperipheral blood, or umbilical cord blood.

126. The method of any of embodiments 115-125, wherein the cell isautologous or allogeneic with respect to a patient to be administeredsaid cell, optionally wherein the patient is a hemoglobinopathy patient,optionally wherein the patient has sickle cell disease or a thalassemia,optionally beta thalassemia.

127. The method of any of embodiments 115-126, wherein:

a) the altering results in an indel at or near a genomic DNA sequencecomplementary to the targeting domain of the one or more gRNA molecules;and/or

b) the altering results in a deletion comprising sequence, e.g.,substantially all the sequence, between a sequence complementary to thetargeting domain of the one or more gRNA molecules (e.g., at least 90%complementary to the gRNA targeting domain, e.g., fully complementary tothe gRNA targeting domain) in the HBG1 promoter region and a sequencecomplementary to the targeting domain of the one or more gRNA molecules(e.g., at least 90% complementary to the gRNA targeting domain, e.g.,fully complementary to the gRNA targeting domain) in the HBG2 promoterregion, optionally wherein the deletion does not comprise a nucleotidedisposed between 5,250,092 and 5,249,833, − strand (hg38).

128. The method of embodiment 127, wherein the indel is an insertion ordeletion of less than about 40 nucleotides, e.g., less than 30nucleotides, e.g., less than 20 nucleotides, e.g., less than 10nucleotides.

129. The method of embodiment 128, wherein the indel is a singlenucleotide deletion.

130. The method of any of embodiments 127-129, wherein the methodresults in a population of cells wherein at least about 15%, e.g., atleast about 17%, e.g., at least about 20%, e.g., at least about 30%,e.g., at least about 40%, e.g., at least about 50%, e.g., at least about55%, e.g., at least about 60%, e.g., at least about 70%, e.g., at leastabout 75%, e.g., at least about 80%, e.g., at least about 85%, e.g., atleast about 90%, e.g., at least about 95%, of the population have beenaltered, e.g., comprise an indel, optionally wherein the indel isselected from an indel listed in Table 2-7, optionally wherein the cellsof the population do not comprise a deletion of a nucleotide disposedbetween 5,250,092 and 5,249,833, − strand (hg38).

131. The method of any of embodiments 115-130, wherein the alteringresults in a cell (e.g., population of cells) that is capable ofdifferentiating into a differentiated cell of an erythroid lineage(e.g., a red blood cell), and wherein said differentiated cell exhibitsan increased level of fetal hemoglobin, e.g., relative to an unalteredcell (e.g., population of cells).

132. The method of any of embodiments 115-131, wherein the alteringresults in a population of cells that is capable of differentiating intoa population of differentiated cells, e.g., a population of cells of anerythroid lineage (e.g., a population of red blood cells), and whereinsaid population of differentiated cells has an increased percentage of Fcells (e.g., at least about 15%, at least about 20%, at least about 25%,at least about 30%, or at least about 40% higher percentage of F cells)e.g., relative to a population of unaltered cells.

133. The method of any of embodiments 115-131, wherein the alteringresults in a cell that is capable of differentiating into adifferentiated cell, e.g., a cell of an erythroid lineage (e.g., a redblood cell), and wherein said differentiated cell produces at leastabout 6 picograms (e.g., at least about 7 picograms, at least about 8picograms, at least about 9 picograms, at least about 10 picograms, orfrom about 8 to about 9 picograms, or from about 9 to about 10picograms) fetal hemoglobin per cell.

134. A cell, altered by the method of any of embodiments 115-133, or acell obtainable by the method of any of embodiments 115-133.

135. A cell, comprising an indel described in Table 7-2, optionallywherein the cell does not comprise a deletion of a nucleotide disposedbetween 5,250,092 and 5,249,833, − strand (hg38).

136. A cell, comprising a first gRNA molecule of any of embodiments1-68, or a composition of any of embodiments 87-104, a nucleic acid ofany of embodiments 105-112, or a vector of any of embodiments 113-114.

137. The cell of embodiment 136, comprising a Cas9 molecule.

138. The cell of embodiment 137, wherein the Cas9 molecule comprises anyof SEQ ID NO: 205, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO:240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243 or SEQ ID NO: 244.

139. The cell of any of embodiments 134-138, wherein the cell comprises,has comprised, or will comprise a second gRNA molecule of any ofembodiments 1-68, or a nucleic acid encoding a second gRNA molecule ofany of embodiments 1-68, wherein the first gRNA molecule and second gRNAmolecule comprise nonidentical targeting domains.

140. The cell of any of embodiments 134-139, wherein expression of fetalhemoglobin is increased in said cell or its progeny (e.g., its erythroidprogeny, e.g., its red blood cell progeny) relative to a cell or itsprogeny of the same cell type that has not been modified to comprise agRNA molecule.

141. The cell of any of embodiments 134-139, wherein the cell is capableof differentiating into a differentiated cell, e.g., a cell of anerythroid lineage (e.g., a red blood cell), and wherein saiddifferentiated cell exhibits an increased level of fetal hemoglobin,e.g., relative to a cell of the same type that has not been modified tocomprise a gRNA molecule.

142. The cell of any of embodiments 140-141, wherein the differentiatedcell (e.g., cell of an erythroid lineage, e.g., red blood cell) producesat least about 6 picograms (e.g., at least about 7 picograms, at leastabout 8 picograms, at least about 9 picograms, at least about 10picograms, or from about 8 to about 9 picograms, or from about 9 toabout 10 picograms) fetal hemoglobin, e.g., relative to a differentiatedcell of the same type that has not been modified to comprise a gRNAmolecule.

143. The cell of any of embodiments 134-142, that has been contactedwith a stem cell expander.

144. The cell of embodiment 143, wherein the stem cell expander is:

a)(1r,4r)—N¹-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamine;

b) methyl4-(3-piperidin-1-ylpropylamino)-9H-pyrimido[4,5-b]indole-7-carboxylate;

c)4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol;

d)(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol;or

e) combinations thereof (e.g., a combination of(1r,4r)—N¹-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamineand(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol).

145. The cell of embodiment 144, wherein the stem cell expander is(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol.

146. A cell, e.g., a cell of any of embodiments 134-145, comprising:

a) an indel at or near a genomic DNA sequence complementary to thetargeting domain of a gRNA molecule of any of embodiments 1-68; and/or

b) a deletion comprising sequence, e.g., substantially all the sequence,between a sequence complementary to the targeting domain of a gRNAmolecule of any of embodiments 1-68 (e.g., at least 90% complementary tothe gRNA targeting domain, e.g., fully complementary to the gRNAtargeting domain) in the HBG1 promoter region and a sequencecomplementary to the targeting domain of a gRNA molecule of any ofembodiments 1-68 (e.g., at least 90% complementary to the gRNA targetingdomain, e.g., fully complementary to the gRNA targeting domain) in theHBG2 promoter region, optionally wherein the deletion, does not comprisea nucleotide disposed between 5,250,092 and 5,249,833, − strand (hg38).

147. The cell of embodiment 146, wherein the indel is an insertion ordeletion of less than about 40 nucleotides, e.g., less than 30nucleotides, e.g., less than 20 nucleotides, e.g., less than 10nucleotides.

148. The cell of any of embodiments 146-147, wherein the indel is asingle nucleotide deletion.

149. The cell of any of embodiments 134-148, wherein the cell is ananimal cell.

150. The cell of embodiment 149, wherein the cell is a mammalian, aprimate, or a human cell.

151. The cell of any of embodiments 134-150, wherein the cell is ahematopoietic stem or progenitor cell (HSPC) (e.g., a population ofHSPCs).

152. The cell of any of embodiments 134-151, wherein the cell is a CD34+cell.

153. The cell of embodiment 152, wherein the cell is a CD34+CD90+ cell.

154. The cell of any of embodiments 134-153, wherein the cell (e.g.population of cells) has been isolated from bone marrow, mobilizedperipheral blood, or umbilical cord blood.

155. The cell of any of embodiments 134-154, wherein the cell isautologous with respect to a patient to be administered said cell,optionally wherein the patient is a hemoglobinopathy patients,optionally wherein the patient has sickle cell disease or a thalassemia,optionally beta thalassemia.

156. The cell of any of embodiments 134-154, wherein the cell isallogeneic with respect to a patient to be administered said cell.

157. A population of cells comprising the cell of any of embodiments134-156.

158. The population of cells of embodiment 157, wherein at least about50%, e.g., at least about 60%, e.g., at least about 70%, e.g., at leastabout 80%, e.g., at least about 90% (e.g., at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about99%) of the cells of the population are a cell according to any one ofembodiments 134-156.

159. The population of cells of any of embodiments 157-158, wherein thepopulation of cells is capable of differentiating into a population ofdifferentiated cells, e.g., a population of cells of an erythroidlineage (e.g., a population of red blood cells), and wherein saidpopulation of differentiated cells has an increased percentage of Fcells (e.g., at least about 15%, at least about 17%, at least about 20%,at least about 25%, at least about 30%, or at least about 40% higherpercentage of F cells) e.g., relative to a population of unmodifiedcells of the same type.

160. The population of cells of embodiment 159, wherein the F cells ofthe population of differentiated cells produce an average of at leastabout 6 picograms (e.g., at least about 7 picograms, at least about 8picograms, at least about 9 picograms, at least about 10 picograms, orfrom about 8 to about 9 picograms, or from about 9 to about 10picograms) fetal hemoglobin per cell.

161. The population of cells of any of embodiments 157-160, comprising:

1) at least 1e6 CD34+ cells/kg body weight of the patient to whom thecells are to be administered;

2) at least 2e6 CD34+ cells/kg body weight of the patient to whom thecells are to be administered;

3) at least 3e6 CD34+ cells/kg body weight of the patient to whom thecells are to be administered;

4) at least 4e6 CD34+ cells/kg body weight of the patient to whom thecells are to be administered; or

5) from 2e6 to 10e6 CD34+ cells/kg body weight of the patient to whomthe cells are to be administered.

162. The population of cells of any of embodiments 157-161, wherein atleast about 40%, e.g., at least about 50%, (e.g., at least about 60%, atleast about 70%, at least about 80%, or at least about 90%) of the cellsof the population are CD34+ cells.

163. The population of cells of embodiment 162, wherein at least about10%, e.g., at least about 15%, e.g., at least about 20%, e.g., at leastabout 30% of the cells of the population are CD34+CD90+ cells.

164. The population of cells of any of embodiments 157-163, wherein thepopulation of cells is derived from umbilical cord blood, peripheralblood (e.g., mobilized peripheral blood), or bone marrow, e.g., isderived from bone marrow.

165. The population of cells of any of embodiments 157-164, wherein thepopulation of cells comprises, e.g., consists of, mammalian cells, e.g.,human cells, optionally wherein the population of cells is obtained froma patient suffering from a hemoglobinopathy, e.g., sickle cell diseaseor a thalassemia, e.g., beta-thalassemia.

166. The population of cells of any of embodiments 157-165, wherein thepopulation of cells is (i) autologous relative to a patient to which itis to be administered, or (ii) allogeneic relative to a patient to whichit is to be administered.

167. The population of cells (e.g., CD34+ cells), e.g., of any ofembodiments 157-165, comprising an indel pattern as described in Table7-2, optionally wherein the indels of an indel pattern described inTable 7-2 are detectible in at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90% or atleast 95% of the cells of the population.

168. A composition comprising a cell of any of embodiments 134-156, orthe population of cells of any of embodiments 157-167.

169. The composition of embodiment 168, comprising a pharmaceuticallyacceptable medium, e.g., a pharmaceutically acceptable medium suitablefor cryopreservation.

170. A method of treating a hemoglobinopathy, comprising administeringto a patient a cell of any of embodiments 134-156, a population of cellsof any of embodiments 157-167, or a composition of any of embodiments168-169.

171. A method of increasing fetal hemoglobin expression in a mammal,comprising administering to a patient a cell of any of embodiments134-156, a population of cells of any of embodiments 157-167, or acomposition of any of embodiments 168-169.

172. The method of embodiment 170, wherein the hemoglobinopathy isbeta-thalassemia or sickle cell disease.

173. A method of preparing a cell (e.g., a population of cells)comprising:

(a) providing a cell (e.g., a population of cells) (e.g., a HSPC (e.g.,a population of HSPCs));

(b) culturing said cell (e.g., said population of cells) ex vivo in acell culture medium comprising a stem cell expander; and

(c) introducing into said cell a first gRNA molecule of any ofembodiments 1-86, a nucleic acid molecule encoding a first gRNA moleculeof any of embodiments 1-86, a composition of any of embodiments 87-104or 168-169, a nucleic acid of any of embodiments 105-112, or a vector ofany of embodiments 113-114.

174. The method of embodiment 173, wherein after said introducing ofstep (c), said cell (e.g., population of cells) is capable ofdifferentiating into a differentiated cell (e.g., population ofdifferentiated cells), e.g., a cell of an erythroid lineage (e.g.,population of cells of an erythroid lineage), e.g., a red blood cell(e.g., a population of red blood cells), and wherein said differentiatedcell (e.g., population of differentiated cells) produces increased fetalhemoglobin, e.g., relative to the same cell which has not been subjectedto step (c).

175. The method of any of embodiments 173-174, wherein the stem cellexpander is:

a)(1r,4r)—N1-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamine;

b) methyl4-(3-piperidin-1-ylpropylamino)-9H-pyrimido[4,5-b]indole-7-carboxylate;

c)4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol;

d)(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol;or

e) combinations thereof (e.g., a combination of(1r,4r)—N1-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamineand(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol).

176. The method of embodiment 175, wherein the stem cell expander is(S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol.

177. The method of any of embodiments 173-176, wherein the cell culturemedium comprises thrombopoietin (Tpo), Flt3 ligand (Flt-3L), and humanstem cell factor (SCF).

178. The method of embodiment 177, wherein the cell culture mediumfurther comprises human interleukin-6 (IL-6).

179. The method of embodiment 177-178, wherein the cell culture mediumcomprises thrombopoietin (Tpo), Flt3 ligand (Flt-3L), and human stemcell factor (SCF) each at a concentration ranging from about 10 ng/mL toabout 1000 ng/mL.

180. The method of embodiment 179, wherein the cell culture mediumcomprises thrombopoietin (Tpo), Flt3 ligand (Flt-3L), and human stemcell factor (SCF) each at a concentration of about 50 ng/mL, e.g, at aconcentration of 50 ng/mL.

181. The method of any of embodiments 178-180, wherein the cell culturemedium comprises human interleukin-6 (IL-6) at a concentration rangingfrom about 10 ng/mL to about 1000 ng/mL.

182. The method of embodiment 181, wherein the cell culture mediumcomprises human interleukin-6 (IL-6) at a concentration of about 50ng/mL, e.g, at a concentration of 50 ng/mL.

183. The method of any of embodiments 173-182, wherein the cell culturemedium comprises a stem cell expander at a concentration ranging fromabout 1 nM to about 1 mM.

184. The method of embodiment 183, wherein the cell culture mediumcomprises a stem cell expander at a concentration ranging from about 1uM to about 100 nM.

185. The method of embodiment 184, wherein the cell culture mediumcomprises a stem cell expander at a concentration ranging from about 500nM to about 750 nM.

186. The method of embodiment 185, wherein the cell culture mediumcomprises a stem cell expander at a concentration of about 500 nM, e.g.,at a concentration of 500 nM.

187. The method of embodiment 186, wherein the cell culture mediumcomprises a stem cell expander at a concentration of about 750 nM, e.g.,at a concentration of 750 nM.

188. The method of any of embodiments 173-187, wherein the culturing ofstep (b) comprises a period of culturing before the introducing of step(c).

189. The method of embodiment 188, wherein the period of culturingbefore the introducing of step (c) is at least 12 hours, e.g., is for aperiod of about 1 day to about 12 days, e.g., is for a period of about 1day to about 6 days, e.g., is for a period of about 1 day to about 3days, e.g., is for a period of about 1 day to about 2 days, e.g., is fora period of about 2 days or for a period of about 1 day.

190. The method of any of embodiments 173-189, wherein the culturing ofstep (b) comprises a period of culturing after the introducing of step(c).

191. The method of embodiment 190, wherein the period of culturing afterthe introducing of step (c) is at least 12 hours, e.g., is for a periodof about 1 day to about 12 days, e.g., is for a period of about 1 day toabout 6 days, e.g., is for a period of about 2 days to about 4 days,e.g., is for a period of about 2 days or is for a period of about 3 daysor is for a period of about 4 days.

192. The method of any of embodiments 173-191, wherein the population ofcells is expanded at least 4-fold, e.g., at least 5-fold, e.g, at least10-fold, e.g., relative to cells which are not cultured according tostep (b).

193. The method of any of embodiments 173-192, wherein the introducingof step (c) comprises an electroporation.

194. The method of embodiment 193, wherein the electroporation comprises1 to 5 pulses, e.g., 1 pulse, and wherein each pulse is at a pulsevoltage ranging from 700 volts to 2000 volts and has a pulse durationranging from 10 ms to 100 ms.

195. The method of embodiment 194, wherein the electroporation comprises1 pulse.

196. The method of any of embodiments 194-195, wherein the pulse voltageranges from 1500 to 1900 volts, e.g., is 1700 volts.

197. The method of any of embodiments 194-196, wherein the pulseduration ranges from 10 ms to 40 ms, e.g., is 20 ms.

198. The method of any of embodiments 173-197, wherein the cell (e.g.,population of cells) provided in step (a) is a human cell (e.g., apopulation of human cells).

199. The method of embodiment 198, wherein the cell (e.g., population ofcells) provided in step (a) is isolated from bone marrow, peripheralblood (e.g., mobilized peripheral blood) or umbilical cord blood.

200. The method of embodiment 199, wherein

(i) the cell (e.g., population of cells) provided in step (a) isisolated from bone marrow, e.g., is isolated from bone marrow of apatient suffering from a hemoglobinopathy, optionally wherein thehemoglobinopathy is sickle cell disease or a thalassemia, optionallywherein the thalassemia is beta thalassemia; or

(ii) the cell (e.g., population of cells) provided in step (a) isisolated from peripheral blood, e.g., is isolated from peripheral bloodof a patient suffering from a hemoglobinopathy, optionally wherein thehemoglobinopathy is sickle cell disease or a thalassemia, optionallywherein the thalassemia is beta thalassemia; optionally wherein theperipheral blood is mobilized peripheral blood, optionally wherein themobilized peripheral blood is mobilized using Plerixafor, G-CSF, or acombination thereof.

201. The method of any of embodiments 173-200, wherein the population ofcells provided in step (a) is enriched for CD34+ cells.

202. The method of any of embodiments 173-201, wherein subsequent to theintroducing of step (c), the cell (e.g., population of cells) iscryopreserved.

203. The method of any of embodiments 173-202, wherein subsequent to theintroducing of step (c), the cell (e.g., population of cells) comprises:

a) an indel at or near a genomic DNA sequence complementary to thetargeting domain of the first gRNA molecule; and/or

b) a deletion comprising sequence, e.g., substantially all the sequence,between a sequence complementary to the targeting domain of the firstgRNA molecule (e.g., at least 90% complementary to the gRNA targetingdomain, e.g., fully complementary to the gRNA targeting domain) in theHBG1 promoter region and a sequence complementary to the targetingdomain of the first gRNA molecule (e.g., at least 90% complementary tothe gRNA targeting domain, e.g., fully complementary to the gRNAtargeting domain) in the HBG2 promoter region, optionally wherein theindel, e.g., deletion, does not comprise a nucleotide disposed between5,250,092 and 5,249,833, − strand (hg38).

204. The method of any of embodiments 173-203, wherein after theintroducing of step (c), at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98% or at least about 99% of the cells of the population ofcells comprise an indel at or near a genomic DNA sequence complementaryto the targeting domain of the first gRNA molecule, optionally whereinno cell of the population comprises a deletion of a nucleotide disposedbetween 5,250,092 and 5,249,833, − strand (hg38).

205. A cell (e.g., population of cells), obtainable by the method of anyof embodiments 173-204.

206. A method of treating a hemoglobinopathy, comprising administeringto a human patient a composition comprising a cell of any of embodiments134-156, a population of cells of any of embodiments 157-167, or a cell(e.g., a population of cells) of embodiment 205.

207. A method of increasing fetal hemoglobin expression in a humanpatient, comprising administering to said human patient a compositioncomprising a cell of any of embodiments 134-156, a population of cellsof any of embodiments 157-167, or a cell (e.g., a population of cells)of embodiment 205.

208. The method of embodiment 206, wherein the hemoglobinopathy isbeta-thalassemia or sickle cell disease.

209. The method of any of embodiments 206-208, wherein the human patientis administered a composition comprising at least about 1e6 cells ofembodiment 205 per kg body weight of the human patient, e.g., at leastabout 1e6 CD34+ cells of embodiment 205 per kg body weight of the humanpatient.

210. The method embodiment 209, wherein the human patient isadministered a composition comprising at least about 2e6 cells ofembodiment 205 per kg body weight of the human patient, e.g., at leastabout 2e6 CD34+ cells of embodiment 205 per kg body weight of the humanpatient.

211. The method embodiment 209, wherein the human patient isadministered a composition comprising from about 2e6 to about 10e6 cellsof embodiment 205 per kg body weight of the human patient, e.g., atleast about 2e6 to about 10e6 CD34+ cells of embodiment 205 per kg bodyweight of the human patient.

212. A gRNA molecule of any of embodiments 1-86, a composition of any ofembodiments 87-114 or 168-169, a nucleic acid of any of embodiments105-112, a vector of any of embodiments 113-114, a cell of any ofembodiments 134-156 or 205, or a population of cells of any ofembodiments 157-167, for use as a medicament.

213. A gRNA molecule of any of embodiments 1-86, a composition of any ofembodiments 87-114 or 168-169, a nucleic acid of any of embodiments105-112, a vector of any of embodiments 113-114, a cell of any ofembodiments 134-156 or 205, or a population of cells of any ofembodiments 157-167, for use in the manufacture of a medicament.

214. A gRNA molecule of any of embodiments 1-86, a composition of any ofembodiments 87-114 or 168-169, a nucleic acid of any of embodiments105-112, a vector of any of embodiments 113-114, a cell of any ofembodiments 134-156 or 205, or a population of cells of any ofembodiments 157-167, for use in the treatment of a disease.

215. A gRNA molecule of any of embodiments 1-86, a composition of any ofembodiments 87-114 or 168-169, a nucleic acid of any of embodiments105-112, a vector of any of embodiments 113-114, a cell of any ofembodiments 134-156 or 205, or a population of cells of any ofembodiments 157-167, for use in the treatment of a disease, wherein thedisease is a hemoglobinopathy.

216. A gRNA molecule of any of embodiments 1-86, a composition of any ofembodiments 87-114 or 168-169, a nucleic acid of any of embodiments105-112, a vector of any of embodiments 113-114, a cell of any ofembodiments 134-156 or 205, or a population of cells of any ofembodiments 157-167, for use in the treatment of a disease, wherein thehemoglobinopathy is beta-thalassemia or sickle cell disease.

EXAMPLES Example 1—Exemplary General Methods

Guide Selection and Design

Initial guide selection was performed in silico using a human referencegenome and user defined genomic regions of interest (e.g., a gene, anexon of a gene, non-coding regulatory region, etc), for identifying PAMsin the regions of interest. For each identified PAM, analyses wereperformed and statistics reported. gRNA molecules were further selectedand rank-ordered based on a number of methods for determining efficiencyand efficacy, e.g., as described herein. This example provides theexperimental details for procedures that can be used to assay the CRISPRsystems, gRNAs and other aspects of the invention described herein. Anymodifications to these general procedures that were employed in aparticular experiment are noted in that example.

Throughout the Examples, in the experiments below, either sgRNAmolecules or dgRNA molecules were used. Unless indicated otherwise,where dgRNA molecules were used, the gRNA includes the following:

crRNA: [SEQ ID NO: 201] [targeting domain] tracr (trRNA): SEQ ID NO: 224

Unless indicated otherwise, in experiments employing a sgRNA molecule,the following sequence was used:

[targeting domain] [SEQ ID NO: 195] UUUU

Next-Generation Sequencing (NGS) and Analysis for On-Target CleavageEfficiency and Indel Formation

To determine the efficiency of editing (e.g., cleaving) the targetlocation in the genome, deep sequencing was utilized to identify thepresence of insertions and deletions introduced by non-homologous endjoining.

In summary PCR primers were designed around the target site, and thegenomic area of interest were PCR amplified in edited and uneditedsamples. Resulting amplicons were converted into Illumina sequencinglibraries and sequenced. Sequencing reads were aligned to the humangenome reference and subjected to variant calling analysis allowing usto the determine sequence variants and their frequency at the targetregion of interest. Data were subjected to various quality filters andknown variants or variants identified only in the unedited samples wereexcluded. The editing percentage was defined as the percentage of allinsertions or deletions events occurring at the on-target site ofinterest (i.e. insertion and deletion reads at the on-target site overthe total number of reads (wild type and mutant reads) at on-targetsite. A detailed description of the NGS analysis process is described inExample 2.1.

RNP Generation

The addition of crRNA and trRNA to Cas9 protein results in the formationof the active Cas9 ribonucleoprotein complex (RNP), which mediatesbinding to the target region specified by the crRNA and specificcleavage of the targeted genomic DNA. This complex was formed by loadingtrRNA and crRNA into Cas9, which is believed to cause conformationalchanges to Cas9 allowing it to bind and cleave dsDNA.

The crRNA and trRNA were separately denatured at 95° C. for 2 minutes,and allowed to come to room temperature. Cas9 protein (10 mg/ml) wasadded to 5×CCE buffer (20 mM HEPES, 100 mM KCl, 5 mM MgCl₂, 1 mM DTT, 5%glycerol), to which trRNA and the various crRNAs were then added (inseparate reactions) and incubated at 37° C. for 10 minutes, therebyforming the active RNP complex. The complex was delivered byelectroporation and other methods into a wide variety of cells,including HEK-293 and CD34+ hematopoietic cells.

Delivery of RNPs to CD34+ HSCs

Cas9 RNPs were Delivered into CD34+ HSCs.

CD34+ HSCs were thawed and cultured (at 500,000 cells/ml) overnight inStemSpan SFEM (StemCell Technologies) media with IL12, SCF, TPO, Flt3Land Pen/Strep added. Roughly 90,000 cells were aliquoted and pelletedper each RNP delivery reaction. The cells were then resuspended in 60 ulP3 nucleofection buffer (Lonza), to which active RNP was subsequentlyadded. The HSCs were then electroporated (e.g., nucleofected usingprogram CA-137 on a Lonza Nucleofector) in triplicate (20uL/electroporation) Immediately following electroporation, StemSpan SFEMmedia (with IL12, SCF, TPO, Flt3L and Pen/Strep) was added to the HSCs,which were cultured for at least 24 hours. HSCs were then harvested andsubjected to T7E1, NGS, and/or surface marker expression analyses.

HSC Functional Assay

CD34+ HSCs may be assayed for stem cell phenotype using known techniquessuch as flow cytometry or the in vitro colony forming assay. By way ofexample, cells were assayed by the in vitro colony forming assay (CFC)using the Methocult H4034 Optimum kit (StemCell Technologies) using themanufacturer's protocol. Briefly, 500-2000 CD34+ cells in <=100 ulvolume are added to 1-1.25 ml methocult. The mixture was vortexedvigorously for 4-5 seconds to mix thoroughly, then allowed to rest atroom temperature for at least 5 minutes. Using a syringe, 1-1.25 ml ofMethoCult+ cells was transferred to a 35 mm dish or well of a 6-wellplate. Colony number and morphology was assessed after 12-14 days as perthe manufacturer's protocol.

In Vivo Xeno-Transplantation

HSCs are functionally defined by their ability to self-renew and formulti-lineage differentiation. This functionality can only be assessedin vivo. The gold-standard for determining human HSC function is throughxeno-transplantation into the NOD-SCID gamma mouse (NSG) that through aseries of mutations is severely immunocompromised and thus can act as arecipient for human cells. HSCs following editing were transplanted intoNSG mice to validate that the induced edit does not impact HSC function.Periodic peripheral blood analysis were used to assess human chimerismand lineage development and secondary transplantation following 20 weekswas used to establish the presence of functional HSCs, as described morefully in these examples.

Example 2.1 Non-Deletional HPFH Region Editing in Hematopoietic Stem andProgenitor Cells (HSPCs) Using CRISPR-Cas9 for De-Repression of FetalGlobin Expression in Adult Erythroid Cells

Methods:

Human CD34⁺ cell culture. Human CD34+ cells were isolated from G-CSFmobilized peripheral blood from adult donors (AllCells) usingimmunoselection (Miltenyi) according to the manufacturer's instructionsand expanded for 4 to 6 days using StemSpan SFEM (StemCell Technologies;Cat no. 09650) supplemented with 50 ng/mL each of thrombopoietin (Tpo,Life Technologies, Cat. #PHC9514), Flt3 ligand (Flt-3L, LifeTechnologies, Cat. #PHC9413), human stem cell factor (SCF, LifeTechnologies, Cat. #PHC2113) and human interleukin-6 (IL-6, LifeTechnologies, Cat. #PHC0063), as well as 1× antibiotic/antimycotic(Gibco, Cat. #10378-016) and 500 nM Compound 4. Throughout this example(including its subexamples), where the protocol indicates that cellswere “expanded,” this medium was used. This medium is also referred toas “stem cell expansion medium,” or “expansion medium” throughout thisexample (including its subexamples).

Assembly of Cas9 and guide RNA ribonucleoprotein (RNP) complexes,preparation of HSPC, and electroporation of RNP into HSPC. Cas9-guideRNA ribonucleoprotein complexes (RNPs) were prepared immediately priorto electroporation. For formation of RNP using dual guide RNAs (dgRNAs),6 μg of each of crRNA (in 4.5 μL) and tracr (in 2.52 μL) are firstdenatured at 95° C. for 2 min in separate tubes and then cooled to roomtemperature. For preparation of Cas9 protein, 12 μg of CAS9 protein (in2 μL) was mixed with 1 μL of 10×CCE buffer (20 mM HEPES, 100 mM KCL, 5mM MgCL2, 5% Glycerol and freshly added 1 mM DTT). Tracr was first mixedwith the Cas9 preparation and incubated at 37° C. for 5 min. The crRNAwas then added to Tracr/CAS9 complexes and incubated for 5 min at 37° C.For the None/control condition, vehicle rather than crRNA was added tothe Tracr/CAS9 complexes. The HSPC were collected by centrifugation andresuspended in P3 buffer + supplement that comes with Lonzaelectroporation kit (Cat #V4XP-3032 Lonza Amaxa P3 primary cell 4-Dnucleofection X Kit S) at a cell density of 2.8×10⁶/mL. The RNP wasmixed with 40 μL of cells by pipetting up and down and incubated at RTfor 2 min. For each replicate 21 μL of the RNP/cell mixture wastransferred into the Lonza Amaxa P3 primary cell 4-D nucleofection X KitS. The electroporation was performed with a Lonza transfection system(4D-Nucleofector X Unit) using protocol CM-137. Duplicate 21 μLelectroporations were performed.

In vitro erythropoiesis and FACS analysis for HbF containing erythroidcells. After electroporation, the cells were immediately transferredinto 250 μL pre-warmed erythroid differentiation medium (EDM) consistingof IMDM (GE Life Sciences, Cat. #SH30228.01). 330 μg/mL humanholo-transferrin (Invitria Cat #777TRF029), 10 μg/mL recombinant humaninsulin (Gibco Cat #A1138211), 2 IU/mL heparin (Sigma, part #H3393), 5%human AB serum (Sigma, Cat #H4522), 2.5 U/mL human erythropoietin(Peprotech #100-064), and 1× antibiotic/antimycotic (Gibco, Cat.#10378-016). During the initial culture period up to day 7, EDM wasfurther supplemented with 1.38 μM hydrocortisone (Sigma H8672), 100ng/mL human SCF (Life Technologies, Cat. #PHC2113), and 5 ng/mL humanIL-3 (Peprotech #10779-598) to make EDM-I. After 4 days, the cellculture was diluted in fresh medium. Cultures were maintained for atotal of 7 days in the culture conditions described above, at which timehalf of the cells were analyzed by intracellular staining for HbFexpression. Briefly, the cells were washed once with PBS, resuspended inLIVE/DEAD® Fixable Violet Dead Cell Stain (ThermoFisher L34963; 1:1000in PBS) and incubated for 30 min. Cell were then washed and stained with1/50 dilutions of anti-CD71-BV711 (Fisher Scientific Company Llc. BD563767) and anti-CD235a-APC (BD 551336) antibodies for 30 min. The cellswere then washed, followed by fixation with fixation buffer (Biolegend,Cat #420801) and permeabilized with 1× intracellular stainingpermeabilization wash buffer (Biolegend, Cat #421002) according to themanufacturers instructions. The cells were then incubated with a 1/40dilution of anti-HbF-PE antibody (Life Technologies, part #MHFH04) in 50μL of 1× intracellular staining perm wash buffer for 20 min at roomtemperature. The cells were washed twice with 0.2 mL of 1× intracellularstaining Perm wash buffer and resuspend in staining buffer and analyzedon an LSRFortessa flow cytometer (BD Biosciences) for HbF expression.The results were analyzed using Flowjo and data were presented as % ofHbF positive cells (F-cells) in the viable CD71 positive erythroid cellpopulation.

From the remaining day 7 cultured cells 80,000 per condition weretransferred into a EDM-II culture medium for further differentiationuntil day 11. EDM-II consists of EDM-I supplemented with only 100 ng/mLSCF. On day 11 cells were counted and 200,000 cells per condition weretransferred into EDM without further supplements. On day 14 cells werestained for analysis of HbF expression similar to day 7 but surfacemarkers were excluded from the staining to prevent aggregation of cells.

Genomic DNA preparation and next generation sequencing (NGS). GenomicDNA was prepared from edited and unedited HSPC at 7 dayspost-electroporation using Quick Extract DNA Extraction Solution(Epicentre Cat #QE09050). To determine editing efficiency and patternsof insertions and deletions (indels), PCR products were generated usingprimers flanking the target sites, which were then subjected to nextgeneration sequencing (NGS) as described in the literature. Percentediting of corresponding sequences in unedited samples (electroporatedwith RNPs consisting of Cas9 and Tracr only) was typically less than 1%and never exceeded 3%.

NGS library preparation and sequencing of amplicons. PCR amplicons werepurified using 1.8× Agencourt AmpureXP beads (Beckman Coulter) followingthe manufactures recommendations. Amplicons were quantified using theQuant-iT PicoGreen dsDNA assay (Life Technologies) following themanufacture's recommendations Illumina sequencing libraries weregenerated using the Nextera DNA Library Prep Kit (Illumina) followingthe manufacture's recommendations with the following changes.Tagmentation was performed in a final volume of 5 ul using 5 ng ofpurified PCR product, 0.15 ul of Nextera tagment enzyme and tagmentationbuffer previously described by Wang et al (PMID: 24071908; incorporatedherein by reference). Tagmented amplicons were then PCR amplified in afinal volume of 50 ul using a final concentration of 0.2 mM dNTP (LifeTechnologies), 0.2 uM Illumina index PCR primers (Integrated DNATechnologies), 1× Phusion DNA polymerase buffer (New England Biolabs)and 1 U of Phusion DNA polymerase (New England Biolabs). PCR cyclingconditions used were as follows: 72° C. for 3 min, 98° C. for 2 min and15 cycles of 98° C. for 10 sec, 63° C. for 30 sec, and 72° C. for 3 min.Sequencing libraries were then purified using 1.0× Agencourt AmpureXPbeads (Beckman Coulter) following the manufactures recommendations.Sequencing libraries were quantified using the Quant-iT PicoGreen dsDNAassay (Life Technologies) following the manufactures recommendations andpooled equimolar for sequencing. Sequencing libraries were sequencedwith 150 base paired-end reads on a MiSeq sequencer following themanufactures recommendations (Illumina) A minimum of a 1000-foldsequencing coverage was generated per amplicon.

NGS sequencing data QC and variant analysis. Using default parameters,the Illumina MiSeq analysis software (MiSeq reporter, version 2.6.2,Illumina) was used to generate amplicon specific FASTQ sequencing datafiles (Cock et al, Nucleic Acids Res. 2010, 38(6):1767-71, PMID:20015970). FASTQ files were then processed through an internallydeveloped variant analysis pipeline consisting of a series of publicdomain software packages joined together using a standard Perl scriptwrapper. The workflow used was divided into five stages.

Stage 1, PCR primer and on- and off-target sequence QC: For both on- andoff-target sites the 20 nucleotide gRNA targeting domain sequence plusPAM sequence and target specific PCR primer sequences (left and rightwithout the additional Illumina sequences) were aligned to the humangenome reference sequence (build GRCh38) using a BLAST search (version2.2.29+, Altschul et al, J Mol Biol., 1990, 215(3):403-10, PMID:2231712). On- and off-target sites with multiple genomic locations wereflagged.

Stage 2, sequencer file decompression: Illumina sequencer generatedFASTQ.GZ files were decompressed to FASTQ files using the gzip script(version 1.3.12) and number of reads per file was calculated. Files withno reads were excluded from further analysis.

Stage 3, sequence read alignment and quality trimming: Sequencing readsin FASTQ files were aligned to the human genome reference sequence(build GRCh38) using the BWA-MEM aligner (version 0.7.4-r385, Li andDurbin, Bioinformatics, 2009, 25(14):1754-60, PMID: 19451168) using‘hard-clipping’ to trim 3′ ends of reads of Illumina sequences and lowquality bases. Resulting aligned reads, in the BAM file format (Li etal, Bioinformatics, 2009 25(16):2078-9, PMID: 19505943), were convertedto FASTQ files using the SAMtools script (version 0.1.19-44428cd, Li etal, Bioinformatics, 2009 25(16):2078-9, PMID: 19505943). FASTQ fileswere then aligned again to the human genome reference sequence (buildGRCh38) using the BWA-MEM aligner, this time without ‘hard-clipping’.

Stage 4, variant (SNP and INDEL) analysis: BAM files of aligned readswere processed using the VarDict variant caller (version 1.0 ‘Cas9aware’ modified by developer ZhongWu Lai, Lai et al, Nucleic Acids Res.,2016, 44(11):e108, PMID: 27060149) with allele frequency detection limitset at >=0.0001 to identify variants (SNPs and indels). The Cas9 awareVarDict caller is based on a public domain package but able to moveambiguous variant calls, generated due to repetitive sequences in thealignment region of the variant events, toward the potential Cas9nuclease cut site in the gRNA targeting domain sequence located 3 bases5′ of the PAM sequence. The SAMtools script was used to calculate readcoverage per sample amplicon to determine whether the on- and off-targetsites were covered at >1000-fold sequence coverage. Sites with<1000-fold sequence coverage were flagged.

Stage 5, dbSNP filtering and treated/untreated differential analysis:Variants identified were filtered for known variants (SNPs and indels)found in dbSNP (build 142, Shery et al, Nucleic Acids Res. 2001,29(1):308-11, PMID: 11125122). Variants in the treated samples werefurther filtered to exclude: 1) variants identified in the uneditedcontrol samples; 2) variants with a VarDict strand bias of 2:1 (whereforward and reverse read counts supporting the reference sequence arebalanced but imbalanced for the non-reference variant call); 3) variantslocated >5 bp either side of the potential Cas9 cut site; 4) singlenucleotide variants.

Results:

We have shown here the surprising result that targeted disruption ofspecific sequences (e.g., by indel creation at or near those sequences)within the HBG1 or HBG2 promoter regions relieves repression of γ-globinexpression, allowing production of the red blood cells containingelevated HbF protein, (cells expressing fetal hemoglobin are sometimesreferred to herein as “F-cells”). The elevated HbF prevents sickling ofthe red blood cells under deoxygenated conditions and will betherapeutic/curative for the patients of both β-thalassemia and SCD.Here, autologous hematopoietic stem cell transplantation (HSCT) with exvivo genome edited HSC from SCD patients was also combined with stemcell expansion enhancing technology, e.g., an aryl hydrocarbon receptor(AHR) inhibitor, e.g., as described in WO2010/059401 (the contents ofwhich are incorporated by reference in their entirety), e.g., Compound4, to improve ex vivo expansion and increase the dose of gene modifiedHSC delivered.

For efficient genome editing via programmable nuclease, Cas9, thesuccessful delivery of guide RNA (gRNA) and Cas9 protein into targetcells and tissues is essential. Here, we show delivery of precomplexedgRNA/Cas9 ribonucleoprotein (RNP) complexes by electroporation leads toefficient and specific genome editing almost immediately after deliveryand are degraded in cells, reducing off-target effects. In contrast, useof plasmid and viral vector systems used to deliver Cas9 results inprolonged expression of the enzyme which may aggravate off-targeteffects associated with the system. Additionally, delivery of RNPs intothe target cells requires no additional tools which would greatlyfacilitate translation of genome editing for therapeutic purposes in theclinic.

Recombinant S. pyogenes Cas9 protein (SEQ ID NO: 236) was purified fromEscherichia coli and complexed with synthetic dual gRNAs (dgRNA) thatconsist of crRNA and tracr to generate ribonucleoprotein (RNP)complexes. The list and sequences of gRNA targeting domains used in thestudy are shown in Table 1. Most of the gRNA sequences have perfecttargets or only 1 or 2 mismatches in both the HBG1 and HBG2 promoterareas. This situation is owed to the duplication of the HBG genes withinthe human beta globin locus. The RNP complexes were electroporated intoCD34+ HSPC via electroporation as described under materials and methods.The cells were expanded prior to the delivery of RNP complexes. Withoutbegin bound by theory, actively dividing cells may facilitate uptake ofRNP complexes delivered by electroporation.

TABLE 4 List of gRNAs targeting the HBG1 and HBG2 promoter region usedin the current study. All gRNA molecules were tested in duplicate in thedgRNA format described above. % % HbF+ (HbF+ % (F % (F HbF+ cells), HbF+% % Guide cells), (F average (F edited % edited % RNA Av Std % averagecells), of cells), HBG1, edited HBG2, edited targeting % CD71+ CD71+ ofstandard replicates standard Average HBG1, Average HBG2, domain day dayreplicates deviation day deviation of standard of standard ID 7) 7) day7 day 7 14 day 14 replicates deviation replicates deviation mock 78.950.21 16.90 0.71 29.15 2.19 n/a n/a n/a n/a g8 73.15 1.20 49.25 0.6466.95 1.48 n/a n/a n/a n/a GCR- 76.05 0.78 50.45 1.63 50.90 1.13 20.774.48 0.40 0.11 0001 GCR- 73.5 1.41 24.50 0.42 33.85 1.91 1.37 0.04 1.540.16 0002 GCR- 72.85 3.32 29.05 1.63 36.00 0.42 2.77 0.67 0.21 0.05 0003GCR- 72.4 0.42 24.60 0.14 32.55 0.21 0.82 0.04 0.82 0.04 0004 GCR- 71.152.76 24.40 0.71 33.00 1.27 0.79 0.02 0.85 0.07 0005 GCR- 76.25 2.3343.40 0.00 43.90 2.40 4.95 0.76 5.92 1.10 0006 GCR- 76.85 1.91 29.701.98 31.25 0.07 3.94 0.81 0.29 0.01 0007 GCR- 70.75 6.15 61.60 2.1255.75 2.90 57.72 4.53 75.09 1.96 0008 GCR- 78.35 0.21 39.95 2.90 41.703.54 11.99 2.60 0.24 0.02 0009 GCR- 73.1 5.52 46.15 2.19 43.30 0.85 5.420.17 9.58 0.63 00010 GCR- 72.2 0.28 43.15 0.07 43.65 1.48 33.62 1.9211.15 1.42 0011 GCR- 69.3 0.42 51.00 0.00 49.05 0.07 44.34 4.16 5.390.08 0012 GCR- 71.95 3.89 23.95 0.21 32.35 0.78 1.02 0.52 0.99 0.50 0013GCR- 76.55 0.64 28.95 0.49 33.65 2.33 5.26 0.37 0.25 0.04 0014 GCR- 76.11.56 30.95 0.64 36.45 0.92 5.58 0.01 0.29 0.05 0015 GCR- 77.35 0.6423.75 0.07 30.90 2.12 0.59 0.08 nd nd 0016 GCR- 77.7 2.97 25.35 0.7829.80 0.42 2.27 0.56 2.00 0.05 0017 GCR- 79.3 0.14 28.05 2.05 33.15 1.773.32 0.74 nd nd 0018 GCR- 79.2 0.99 28.30 1.70 34.65 0.92 4.47 0.14 6.171.19 0019 GCR- 78.7 0.57 25.30 1.56 31.05 0.07 1.25 0.64 nd nd 0020 GCR-76.05 0.07 24.75 0.64 30.70 2.69 1.09 0.01 1.41 0.20 0021 GCR- 75.751.06 25.00 1.56 32.25 0.92 0.24 0.02 2.01 0.16 0022 GCR- 76.95 2.7623.00 0.57 31.80 0.85 0.31 0.05 nd nd 0023 GCR- 77.7 1.56 22.90 0.2830.50 1.41 0.47 0.25 nd nd 0024 GCR- 77 0.71 26.70 0.85 32.80 0.71 1.590.01 nd nd 0025 GCR- 76.85 2.05 24.15 0.49 30.95 0.35 0.95 0.18 1.430.11 0026 GCR- 78.65 0.35 25.80 0.00 29.00 1.13 nd nd 0.78 0.01 0027GCR- 79 2.97 50.60 0.71 47.90 0.85 24.90 2.02 nd nd 0028 GCR- 74.7 0.9928.90 1.13 32.50 1.70 0.19 0.10 4.09 1.00 0029 GCR- 77 3.25 24.95 0.9232.20 0.57 0.96 0.08 nd nd 0030 GCR- 79 0.99 30.40 0.71 36.00 1.70 2.870.93 2.98 0.84 0031 GCR- 78.55 0.21 26.30 0.28 33.00 0.71 4.25 0.56 4.610.66 0032 GCR- 78.75 0.92 27.20 1.56 32.95 1.91 3.00 0.68 3.58 0.28 0033GCR- 80.05 0.49 50.55 1.48 50.35 1.48 6.31 0.24 12.68 0.64 0034 GCR-79.75 0.35 23.30 0.00 31.30 0.71 3.68 0.16 nd nd 0035 GCR- 79.7 0.5723.50 0.28 30.95 0.07 0.71 0.21 1.51 0.36 0036 GCR- 74.9 2.26 34.85 0.4934.55 0.64 4.30 0.67 7.95 0.83 0037 GCR- 77 0.14 25.20 0.28 30.55 2.191.61 0.13 nd nd 0038 GCR- 75.35 1.06 25.50 0.28 34.40 0.99 2.66 0.620.43 0.01 0039 GCR- 76.35 1.06 28.20 0.71 33.50 0.57 2.66 0.51 2.61 0.210040 GCR- 74.8 3.68 24.30 0.99 33.80 0.71 0.90 0.14 nd nd 0041 GCR- 73.64.10 26.45 0.49 33.65 2.76 1.75 0.22 2.43 0.13 0042 GCR- 70.4 0.00 25.450.21 33.25 0.21 1.17 0.02 1.66 0.17 0043 GCR- 74.7 0.00 26.10 0.85 34.252.19 2.40 0.14 3.65 0.01 0044 GCR- 75.75 1.91 41.30 1.70 41.55 1.34 7.460.71 11.75 2.79 0045 GCR- 77.85 1.91 41.30 1.70 42.40 0.71 1.69 0.0313.10 0.15 0046 GCR- 74.6 2.40 49.15 4.03 39.90 2.12 24.89 6.43 45.5910.58 0047 GCR- 76.85 4.88 60.75 1.77 52.95 2.62 28.49 10.88 47.10 15.490048 GCR- 78.05 0.21 34.50 0.57 37.45 1.77 3.39 0.55 nd nd 0049 GCR-76.25 6.72 45.90 2.12 41.20 0.57 21.36 8.74 30.16 1.60 0050 GCR- 81.552.19 54.55 0.92 51.35 1.77 25.09 0.33 77.84 1.39 0051 GCR- 80.6 0.1437.25 1.77 42.35 0.35 0.31 0.04 20.52 1.70 0052 GCR- 80.35 0.64 39.751.20 43.45 1.63 6.81 0.57 10.47 2.01 0053 GCR- 81.05 0.21 44.55 0.7846.55 1.48 0.48 0.10 23.91 2.87 0054 GCR- 81.4 0.71 22.95 0.35 31.700.42 1.79 0.62 2.40 0.47 0055 GCR- 81.1 0.85 31.70 0.14 37.00 0.28 2.630.46 5.24 0.66 0056 GCR- 72.4 6.08 25.25 0.78 30.25 1.20 0.88 0.56 nd nd0057 GCR- 70.95 3.04 40.00 0.71 39.25 0.49 0.54 0.13 17.84 1.33 0058GCR- 73.85 2.76 29.75 0.64 33.85 0.07 3.36 0.18 5.56 0.91 0059 GCR- 678.20 24.70 0.14 30.95 0.07 0.99 0.06 1.06 0.38 0060 GCR- 71.1 3.82 30.050.49 35.80 0.42 2.91 0.67 2.07 nd 0061 GCR- 74.9 4.67 43.95 3.46 43.602.26 7.77 1.89 13.23 2.01 0062 GCR- 77.05 2.62 42.20 1.13 43.80 1.1311.32 0.84 17.89 1.55 0063 GCR- 78.85 0.21 21.85 0.92 31.60 1.41 0.830.12 0.81 0.08 0064 GCR- 78.15 0.21 26.35 2.05 34.75 0.49 nd nd nd nd0065 GCR- 78.85 1.77 23.85 0.07 31.55 0.64 4.86 0.18 4.89 0.08 0066 GCR-69 7.07 62.75 0.92 53.85 2.19 16.54 4.20 29.61 7.91 0067 GCR- 78.1 1.8427.75 1.77 34.40 0.71 3.77 0.12 0.39 0.07 0068 GCR- 79.15 2.19 30.000.14 35.40 1.56 4.28 0.10 6.23 1.40 0069 GCR- 77.9 0.14 30.70 0.42 36.051.20 3.47 0.56 nd nd 0070 GCR- 79.2 0.42 36.80 2.55 39.80 2.12 67.168.10 nd nd 0071 GCR- 79.25 1.20 26.20 0.28 34.60 1.13 2.89 0.88 3.480.64 0072

The genome edited and unedited HSPC were analyzed by flow cytometry forexpression levels of fetal globin and the erythroid cell surface markertransferrin receptor (CD71) using antibodies conjugated to fluorescentdyes. The live cells were identified and gated by exclusion of Live DeadViolet. Genome editing did not adversely affect erythroiddifferentiation as the cultured cells showed percentages of CD71+ cellsconsistent with erythroblasts similar to uneditied cells. Delivery ofthese gRNA RNPs to HSPCs resulted in an increased percentage of progenyerythroid cells containing HbF (up to 62.75%) compared to mockelectroporated cells (16.9%) at day 7 following electroporation (Table4). Additional evaluation of HbF induction levels on day 14 confirmedhigh induction levels for the best performing dgRNA sequences (table 4),although background HbF levels detected in controls were higher comparedto day 7. Induction of HbF positive cells by dgRNA including thetargeting domain of g8 targeting exon 2 of BCL11A was also observed inparallel, and several of the gRNAs to the HBG1 or HBG2 regions resultedin higher % F cell levels than g8. PCR products from genomic DNA of theHBG1 and HBG2 promoter region isolated on day 7 after electroporationwere also subjected to next generation sequencing (NGS) to determine thepercentage of edited alleles in the cell population. High genome editingpercentages at the HBG1 and HBG2 promoter region was observed in many ofthe cell cultures electroporated with RNPs containing Cas9, crRNA of thegiven targeting domain and Tracr (Table 4, FIG. 1), but not in controlcells with no targeting domain delivered (RNPs containing only Cas9 andTmcr). In particular, the dgRNA treatments that resulted in greater than17% HbF+ cells above mock transfected control background on Day 7 had arange of 5.92% to 77.84% edited alleles in either the HBG1 or HBG2target loci (Table 4, FIG. 2 and FIG. 3). Some guides with selectivespecificity for either the HBG1 or HBG2 promoter area showedpreferential editing at the locus they are specific for (for exampleGCR-0001, GCR-0011 and GCR-0012 edit HBG1 more efficiently and GCR-0034,GCR-0046, GCR-0051, GCR-0052, GCR-0054 and GCR-0058 edit HBG2 moreefficiently). A notable and surprising exception to this correlation isGCR-0008 which, although specific to a target sequence of the HBG1locus, edits both HBG1 and HBG2 loci efficiently and also results inhigh HbF induction. GCR-0008 has a single mismatch with the targetsequence in the HBG2 locus suggesting that efficient off-target editingoccurs at this site. The target sequence of some of the 72 gRNAs testedmap to regions overlapping or near several annotated human mutationsassociated with hereditary persistence of fetal hemoglobin (HPFH)expression as well as to known binding sites for transcription factorsin the proximal promoter areas of both HBG1 and HBG2 (FIGS. 4A-4D andFIGS. 5A-5D). Surprisingly, a cluster of the best performing gRNAstarget sequences outside of these known areas of promoter function inthe HBG1 and HBG2 genes (e.g., map to chr11:5,250,094-5,250,237; hg38and chr11:5,255,022-5,255,164; hg38 (FIGS. 4A-4D and FIGS. 5A-5D),respectively). These gRNAs include GCR-0001, GCR-0006, GCR-0008,GCR-0009, GCR-0010, GCR-0011, GCR-0012, GCR-0034, GCR-0046, GCR-0048,GCR-0051, GCR-0054, GCR-0058 and GCR-0067. Another well performing gRNA(GCR-0028) targets a transcription factor binding site (TATA box) in theproximal promoter area both of HBG1 and HBG2 that is not associated withnon-deletional HPFH mutations (chr11:5,249,833-5,249,927; hg38 andchr11:5,254,738-5,254,851; hg38 (FIGS. 4A-4D and FIGS. 5A-5D),respectively).

Example 2.2—Gamma Globin Promoter Region Editing in HSPCs forDe-Repression of Fetal Globin Expression in Adult ErythroidCell—Evaluation at Select Sites Using CRISPR-Cas9 with sgRNA Format

Methods:

Methods are as in Example 2.1, with the following modifications.

Human CD34⁺ cell culture Human CD34+ cells were derived from adulthealthy donor bone marrow (Lonza catalog #2M-101D). Cells were thawedthen expanded for 6 days.

Assembly of Cas9 and guide RNA ribonucleoprotein (RNP) complexes,preparation of HSPC, and electroporation of RNP into HSPC. For formationof RNP using single guide RNAs (sgRNAs), 12 μg of each of sgRNA, 12 μgof CAS9 protein and 1 μL of 10×CCE buffer (20 mM HEPES, 100 mM KCL, 5 mMMgCL2, 5% Glycerol and freshly added 1 mM DTT) were combined in a totalvolume of 10 ul, then incubated at 37° C. for 5 min. For theNone/control condition, vehicle rather than sgRNA was added. Celldensity in P3 buffer + supplement was 3.9×10⁶/mL.

In vitro erythropoiesis and FACS analysis for HbF containing erythroidcells. On day 11 cells were transferred into EDM without furthersupplements. On day 14 and day 18 cells were pelleted and resuspended infresh EDM without further supplements. An aliquot of cells was taken foranalysis of HbF expression at day 14 and day 21. Cells were stainedsimilar to day 7, but surface markers were excluded from the staining toprevent aggregation of cells and anti-HbF antibody was used at 1:20dilution.

Genomic DNA preparation and next generation sequencing (NGS). GenomicDNA was prepared from edited and unedited HSPC at 3 dayspost-electroporation using Quick Extract DNA Extraction Solution(Epicentre Cat #QE09050). NGS analysis described below showed nosignificant editing in control samples electroporated with Cas9 alone.

NGS library preparation and sequencing of amplicons was carried out asdescribed in Example 2.1. NGS sequencing data QC and variant analysiswas performed as described in Example 2.1.

Results:

We have shown here that the targeted disruption of specific sequenceswithin the HBG1 or HBG2 promoter regions leading to the production ofF-cells can also be accomplished by utilizing gRNA of the sgRNA format.

TABLE 5 List of select gRNAs targeting the HBG1 and HBG2 promoter regionused in the current study. All gRNA molecules were tested in duplicatein the sgRNA format described above. % % % (HbF+ % HbF+ % HbF+ % (F HbF+(F HbF+ (F HbF+ cells), (F cells), (F % % Guide cells), (F averagecells), average cells), edited % edited % RNA average cells), ofstandard of standard HBG1, edited HBG2, edited targeting of standardreplicates deviation replicates deviation Average HBG1, Average HBG2,domain replicates deviation day day day day of standard of standard IDday 7 day 7 14 14 21 21 replicates deviation replicates deviationNone/control 39.0 1.8 32.7 0.6 14.4 4.9 n/a n/a n/a n/a GCR- 60.8 2.549.2 0.4 20.5 7.6 15.8 3.0 0.0 0.0 0001 GCR- 67.1 0.8 55.2 2.3 25.6 9.355.2 2.0 40.9 2.8 0008 GCR- 61.3 0.8 52.3 1.0 14.0 0.8 4.8 0.8 3.0 0.00010 GCR- 58.7 1.6 49.2 0.1 14.1 2.1 nd nd nd nd 0028 GCR- 52.6 1.6 41.24.1 16.7 3.3 nd nd nd nd 0047 GCR- 64.3 2.3 57.5 2.3 28.8 11.0 53.7 5.357.2 5.3 0048 GCR- 65.3 3.0 59.1 2.0 20.6 5.7 27.3 0.3 50.6 3.8 0051GCR- 71.7 3.3 73.7 3.3 26.2 2.6 11.9 4.2 28.0 6.1 0053 GCR- 67.3 2.061.4 4.0 23.4 3.5 nd nd 18.4 1.9 0054 GCR- 61.1 5.2 54.8 1.1 16.7 1.72.5 1.8 3.9 2.7 0062 GCR- 63.6 3.7 58.0 1.7 24.6 1.3 16.4 0.2 29.9 2.90063 GCR- 74.6 0.4 70.4 3.7 36.6 0.6 23.7 0.0 37.5 4.4 0067

Adult bone marrow-derived HSPC were electroporated with RNP complexesformed from recombinant S. pyogenes Cas9 protein (SEQ ID NO: 236) andthe indicated gRNA of the sgRNA format. The resulting genome edited andunedited HSPC were analyzed by flow cytometry for expression levels offetal globin and the erythroid cell surface marker transferrin receptor(CD71) using antibodies conjugated to fluorescent dyes. The live cellswere identified and gated by exclusion of Live Dead Violet. Delivery ofthese gRNA RNPs to HSPCs resulted in an increased percentage of progenyerythroid cells containing HbF (up to 74.6%) compared to mockelectroporated cells (39.0%) at day 7 following electroporation (Table5). Additional evaluation of HbF induction levels on day 14 and day 21confirmed high induction levels for the best performing sgRNA sequences(Table 5). PCR products from genomic DNA of the HBG1 and HBG2 promoterregion isolated on day 3 after electroporation were also subjected tonext generation sequencing (NGS) to determine the percentage of editedalleles in the cell population. High genome editing percentages(excluding large deletions) at the HBG1 and HBG2 promoter region wasobserved in many of the cell cultures electroporated with RNPscontaining Cas9 and sgRNA of the given targeting domain (Table 5), butnot in control cells with no sgRNA delivered (Cas9 only).

Select targeting sites with a >17% increase in HbF+ erythroid cells atday 7 in Example 2.1 were included in this study. The experimentaldesign differed in two significant ways from that previously describedin Example 2.1: the gRNA format (sgRNA rather than dgRNA) and the HSPCsource (different donor and bone marrow-derived rather than mobilizedperipheral blood-derived). Without being bound by theory, thedifferences in baseline percentages of HbF+ cells in unedited cellcultures between studies may be caused by the inherent differencesbetween HSPC sources. Despite these differences, all targeting siteswith the exception of GCR-47 remained associated with a >17% increase inHbF+ erythroid cells at day 7 (FIG. 7). This increase was maintained outto day 21 for GCR-0067 (FIG. 7). Additionally, indel formation of >25%at HBG1, HBG2 or both was observed with GCR-0008, GCR-0048, GCR-0051,GCR-0053, GCR-0063 and GCR-0067 (Table 5). As with the dgRNA format(Table 4), GCR-0008 in the sgRNA format was associated with efficientediting at both the on-target HBG1 site and the off-target HBG2 site(Table 5). It was undetermined whether the lower HbF+ cell inductionwith GCR-0047 in this study was associated with reduced editing.Notably, day 7 and day 14 HbF+ cell induction of >17% was associatedwith targeting sequences outside of the known areas of promoter functionin the HBG1 and HBG2 genes (e.g., map to chr11:5,250,094-5,250,237; hg38and chr11:5,255,022-5,255,164; hg38 (FIGS. 4A-4D and FIGS. 5A-5D),respectively), specifically GCR-0001, GCR-0008, GCR-0010, GCR-0048,GCR-0051, GCR-0054 and GCR-0067 (FIG. 7). Of these, GCR-0008, GCR-0048and GCR-0067 also had a >10% increase in HbF+ cells at day 21 and >25%indels at HBG1, HBG2 or both (Table 5 and FIG. 7).

Example 2.3—Gamma Globin Promoter Region Editing in HSPCs forDe-Repression of Fetal Globin Expression in Adult ErythroidCells—Additional Analysis of Editing Patterns Using CRISPR-Cas9 withsgRNA Format

Methods:

Methods are as in Example 2.1, with the following modifications.

Human CD34⁺ cell culture. Human CD34+ cells were derived from bonemarrow from adult healthy donors (Lonza catalog #2M-101D and Hemacarecatalog #BM34-C). Cells were thawed then expanded for 6 days.

Assembly of Cas9 and guide RNA ribonucleoprotein (RNP) complexes,preparation of HSPC, and electroporation of RNP into HSPC. For formationof RNP using single guide RNAs (sgRNAs), 12 μg of each of sgRNA, 12 μgof CAS9 protein and 1 μL of 10×CCE buffer (20 mM HEPES, 100 mM KCL, 5 mMMgCL2, 5% Glycerol and freshly added 1 mM DTT) were combined in a totalvolume of 10 ul, then incubated at 37° C. for 5 min. For theNone/control condition, vehicle rather than sgRNA was added. Celldensity in P3 buffer + supplement was 3.9×10⁶/mL.

In vitro erythropoiesis and FACS analysis for HbF containing erythroidcells. On day 11 cells were transferred into EDM without furthersupplements. On day 14 and day 18 cells were pelleted and resuspended infresh EDM without further supplements. An aliquot of cells was taken foranalysis of HbF expression at day 14 and day 21. Cells were stainedsimilar to day 7, but surface markers were excluded from the staining toprevent aggregation of cells and anti-HbF antibody was used at 1:20dilution.

Genomic DNA preparation and next generation sequencing (NGS). GenomicDNA was prepared from edited and unedited HSPC at 3 dayspost-electroporation using Quick Extract DNA Extraction Solution(Epicentre Cat #QE09050). NGS analysis described below showed nosignificant editing in control samples electroporated with Cas9 alone.

NGS library preparation and sequencing of amplicons was carried out asdescribed in Example 2.1. NGS sequencing data QC and variant analysiswas performed as described in Example 2.1.

Non-quantitative PCR for detection of inversions and large deletions.The following primer sets were used to amplify gDNA in the region ofHBG1 and HBG2. P1: forward primer 5′-TGCTGAGATGAAACAGGCGT-3′ (SEQ ID NO:257), reverse primer 5′-TTAGGCATCCACAAGGGCTG-3′ (SEQ ID NO: 258),expected ˜2.8 kb product for deletion between HBG1 and HBG2target/off-target sites, expected ˜7.7 kb product for inversion betweenHBG1 and HBG2 target/off-target sites, expected ˜7.7 kb product for nolarge deletion or inversion (unedited or smaller indels at HBG1 and/orHBG2 target/off-target sites, individually). P2: forward primer5′-GCTCTACAAATGGAACCCAACC-3′ (SEQ ID NO: 259), reverse primer5′-CTGCTCTGATCTCTAACACCTCA-3′ (SEQ ID NO: 260), no product expected fordeletion between HBG1 and HBG2 target/off-target sites, no productexpected for inversion between HBG1 and HBG2 target/off-target sites,expected ˜3.8 kb product for no large deletion or inversion (unedited orsmaller indels at HBG1 and/or HBG2 target/off-target sites,individually). P3: forward primer 5′-GAAGATACAGCTTGCCTCCGA-3′ (SEQ IDNO: 261), reverse primer 5′-TTGCTGAGATGAAACAGGCGT-3′ (SEQ ID NO: 262),no product expected for deletion between HBG1 and HBG2 target/off-targetsites, expected ˜1.75 kb product for inversion between HBG1 and HBG2target/off-target sites, no product expected for no large deletion orinversion (unedited or smaller indels at HBG1 and/or HBG2target/off-target sites, individually). PCR products were visualized onan agarose gel along with a reference ladder containing 0.5, 1, 1.5, 2,3, 4, 5, 6, 8 and 10 kb bands (L; New England Biolabs catalog #N3232L).Select products were isolated as indicated and subjected to NGS asdescribed in Example 2.1.

Quantification of large deletions. Large deletions between targetingsites at the HBG1 and HBG2 promotors were quantified by digital dropletPCR (ddPCR) in non-competitive assay format for copy numberdetermination according to manufacturer's recommendations. Briefly, gDNAwas combined with ddPCR SuperMix for Probes (no dUTPs) (BioRad Cat#1863024), HindIII-HF restriction enzyme (NEB Cat #R3104S) and eachprimer probe mix, transferred to a DG8 cartridge along with DropletGeneration Oil for Probes (BioRad Cat #1863005) and droplets generatedwith a QX200 droplet generator (BioRad). Droplets were subject to PCR ona C1000 Touch Thermal Cycler with 96-Deep Well Reaction Module (BioRad),followed by detection with QX200 droplet reader (BioRad). Copies per ulwas determined by analysis with QuantaSoft software (BioRad). A customprimer probe set (Life Technologies Cat #APZW76R, PN4331348, Forwardprimer: ACGGATAAGTAGATATTGAGGTAAGC (SEQ ID NO: 263), Reverse primer:GTCTCTTTCAGTTAGCAGTGG (SEQ ID NO: 264), FAM TaqMan Probe:ACTGCGCTGAAACTGTGGCTTTATAG (SEQ ID NO: 265)) used to amplify gDNA withinthe HBG1-HBG2 intergenic region. A TaqMan Copy Number Reference Assay,human, RNase P (Thermo Fisher cat #4403326) was used as a referenceamplicon. Copies per ul for HBG1-HBG2 and RNase P amplicons were withinthe manufacturer's reported linear range. Percent deletion was reportedas 100% times 1 minus the ratio of copies per ul for HBG1-HBG2 and RNaseP amplicons. Unedited control samples had calculated percent deletionsof 2.4%, which may reflect background in the assay.

Results:

We have shown here that the targeted disruption of specific sequenceswithin the HBG1 and HBG2 promoter regions leading to the production ofF-cells is associated with both indels at the HBG1 or HBG2 target oroff-target site, as well as deletions and inversions of the interveningregion. NGS analysis of amplicons confirms this observation.

TABLE 6 List of select gRNAs targeting the HBG1 and HBG2 promoter regionused in the current study to edit cells from the first independentdonor. All gRNA molecules were tested in duplicate in the sgRNA formatdescribed above. % % % % % HbF+ % HbF+ HbF+ HbF+ HbF+ (F HbF+ (F (F (F(F % % Guide cells), (F cells), cells), cells), cells), edited % edited% RNA average cells), average standard average standard HBG1, editedHBG2, edited targeting of standard of deviation of deviation AverageHBG1, Average HBG2, domain replicates deviation replicates dayreplicates day of standard of standard ID day 7 day 7 day 14 14 day 2121 replicates deviation replicates deviation none/control 37.6 3.0 17.10.4 32.4 5.9 n/a n/a n/a n/a GCR- 61.3 1.1 45.6 0.4 75.1 2.1 59.1 3.91.0 0.4 0001 GCR- 62.9 1.3 42.8 2.1 67.3 1.3 86.3 6.2 85.6 5.5 0008 GCR-69.5 3.0 53.4 0.9 75.0 0.4 19.5 6.2 25.1 1.0 0010 GCR- 59.2 4.2 43.8 0.866.2 3.6 73.5 9.2 80.4 3.6 0048 GCR- 61.9 0.4 49.7 1.7 70.1 6.2 53.0 0.586.4 0.6 0051 GCR- 72.2 1.8 58.1 1.7 77.4 4.2 68.8 1.2 77.5 1.6 0067

TABLE 7 List of select gRNAs targeting the HBG1 and HBG2 promoter regionused in the current study to edit cells from the second independentdonor. All gRNA molecules were tested in duplicate in the sgRNA formatdescribed above. % % % % % HbF+ % HbF+ HbF+ HbF+ HbF+ (F HbF+ (F (F (F(F % % Guide cells), (F cells), cells), cells), cells), edited % edited% RNA average cells), average standard average standard HBG1, editedHBG2, edited targeting of standard of deviation of deviation AverageHBG1, Average HBG2, domain replicates deviation replicates dayreplicates day of standard of standard ID day 7 day 7 day 14 14 day 2121 replicates deviation replicates deviation none/control 39.3 0.2 23.10.8 52.7 0.3 n/a n/a n/a n/a GCR- 58.8 0.7 44.7 3.0 80.2 0.8 62.7 10.0  1.0 0.0 0001 GCR- 61.6 2.2 44.4 0.7 75.0 0.5 92.1 5.7 86.4 5.5 0008GCR- 65.5 2.5 47.7 3.0 79.4 0.0 17.7 n/a 28.5 9.0 0010 GCR- 60.5 0.346.1 3.3 79.3 1.2 68.4 6.6 76.9 9.3 0048 GCR- 64.5 1.7 47.1 4.2 78.1 0.462.7 7.8 86.4 3.0 0051 GCR- 68.8 4.6 54.9 0.4 84.5 3.1 77.4 8.7 78.0 4.20067

Adult bone marrow-derived HSPC from 2 independent donors wereelectroporated with RNP complexes formed from recombinant S. pyogenesCas9 protein (SEQ ID NO: 236) and the indicated gRNA of the sgRNAformat. Evaluated sgRNAs had targeting sequences outside of the knownareas of promoter function in the HBG1 and HBG2 genes (e.g., map tochr11:5,250,094-5,250,237; hg38 and chr11:5,255,022-5,255,164; hg38(FIGS. 4A-4D and FIGS. 5A-5D), respectively). The resulting genomeedited and unedited HSPC were analyzed by flow cytometry for expressionlevels of fetal globin and the erythroid cell surface marker transferrinreceptor (CD71) using antibodies conjugated to fluorescent dyes. Thelive cells were identified and gated by exclusion of Live Dead Violet.Delivery of these gRNA RNPs to HSPCs resulted in an increased percentageof progeny erythroid cells containing HbF compared to mockelectroporated cells at days 7, 14 and 21 following electroporation(Tables 6 and 7). All included targeting sites were associated witha >17% increase in HbF+ erythroid cells from both donors at alltimepoints—days 7, 14 and 21 (FIG. 8). PCR products from genomic DNA ofthe HBG1 and HBG2 promoter region isolated on day 3 afterelectroporation were also subjected to next generation sequencing (NGS)to determine the percentage of edited alleles in the cell population.High genome editing percentages at both the HBG1 and HBG2 promoterregion (53% to 92% indels) was observed in cell cultures from bothdonors electroporated with RNPs containing Cas9 and sgRNAs withtargeting domains GCR-0008, GCR-0048, GCR-0051 and GCR-0067 (Tables 6and 7), but not in control cells with no sgRNA delivered (Cas9 only).Targeting domain GCR-0010 was associated with reduced but sizableediting percentages at both the HBG1 and HBG2 promotor region (17% to28% indels) in cell cultures from both donors; whereas, GCR-0001 wasassociated with efficient and selective editing at HBG1 (59% and 62%)compared with HBG2 (1% and 1%) in the two donors (Tables 6 and 7).

The GCR-0048 and GCR-0067 targeting domains are present at both HBG1 andHBG2, and the other targeting domains are present at either HBG1(GCR-0001, GCR-0008 and GCR-0010) or HBG2 (GCR-0051) with a potentialmismatched off-target site at the other promotor. Simultaneous cleavageat both the HBG1 and HBG2 target/off-target sites has the potential toresult in deletion and/or inversion of the intervening 4.9 kb genomicsequence; herein sometimes also referred to as ‘4.9 kb’ or HBG1-HBG2′ or‘large’ inversion or deletion. Thus, a set of three different PCRreactions was used to detect the presence of genomes with deletion orinversion of this region. The P2 reaction was designed so that genomicsequences without the HBG1-HBG2 deletion or inversion would be amplifiedwith a resulting 3.8 kb product, whereas sequences with the HBG1-HBG2deletion or inversion would not be amplified. A band approximately ofthis size was detected in all samples (FIG. 9), consistent with thedetection of smaller indels at each individual HBG1 or HBG2 targetingsite (Tables 6 and 7). The P3 reaction was designed so that HBG1-HBG2inverted genomic sequences would be amplified with a resulting 1.7 kbproduct, whereas sequences without this inversion or with/without theHBG1-HBG2 deletion would not be amplified. A band approximately of thissize was detected in cultures electroporated with RNPs containing Cas9and sgRNAs with the indicated targeting domains, but was undetectable inunedited control cultures (FIG. 9). Finally, the P1 reaction wasdesigned to span both the HBG1 and HBG2 targeting region, so thatamplification of sequences without the HBG1-HBG2 deletion as well asthose with the HBG1-HBG2 region inverted would both produce a 7.7 kbproduct, while amplification of sequences containing the HBG1-HBG2deletion would result in a 2.8 kb product. Indeed, unedited controlsamples had a prominent upper band at approximately 8 kb and a faint,potentially non-specific band at approximately 2.8 kb. In contrast,cultures electroporated with RNPs containing Cas9 and sgRNAs with theindicated targeting domains had a prominent band at approximately 2.8 kband a faint band at approximately 8 kb (FIG. 9). DNA from each bandindicated with an asterix was isolated and subject to next-generationsequencing to confirm its identity as amplification of the predictedsequence (with or without the HBG1-HBG2 inversion or deletion, asdescribed).

For further sequence characterization of the region spanning the HBG1and HBG2 editing sites, a fourth long range PCR condition (P4) wasdeveloped to amplify a 6192 bp region encompassing the HBG1 and HBG2editing sites for gRNA GCR-0001, GCR-0008, GCR-0010, GCR-0048, GCR-0051and GCR-0067. Amplification of genome edited samples results in twoamplicon sizes (1.2 kb and a 6.2 kb), while unedited samples onlygenerate one amplicon size (6.2 kb). Sequence analysis of the 6.2 kb and1.2 kb amplicons shows that the 1.2 kb amplicons are a result of 4.9 kbdeletions between the HBG1 and HBG2 cut sites, while the 6.2 kb ampliconconsist of wildtype and various indel alleles at the HBG1 and HBG2editing sites. Sequencing analysis also shows a low level of inversionsbetween the HBG1 and HBG2 cut sites where the sequence excised betweenthe two sites has been reincorporated into the genome in the oppositedirection. This analysis is not quantitative, but the inversion islikely very rare as it is only detected in a small percentage (<1%) ofsequencing reads spanning the HBG1 and HBG2 cut sites.

Together these results suggest that, in addition to indels at eitherHBG1 or HBG2 target/off-target site, cultures electroporated with RNPscontaining Cas9 and sgRNAs with the indicated targeting domains containedited alleles with deletion or inversion of the intervening HBG1-HBG2region. Thus, a given allele post-editing could be an HBG1-HBG1inversion, an HBG1-HBG2 deletion, a indel localized to the HBG1 site, anindel localized to the HBG2 site, or an indel localized to the HBG1 sitealong with an indel localized to the HBG2 site without modification ofthe intervening region. The most common on-target editing repair patternvariants generated by editing with gRNAs GCR-0001, GCR-0008, GCR-0010,GCR-0048, GCR-0051 and GCR-0067 are shown in Table 7-2. The variantsshown are localized indels generated at the HBG1 and HBG2 loci and large4.9 kb deletions caused by the excision of the sequence between the HBG1and HBG2 cut sites. The localized indels were characterized using PCRand NGS analysis as described in Example 2.1. A quantitative ddPCR assaywas developed to determine the frequency of alleles with the large 4.9kb deletion of the intervening HBG1 to HBG2 region (FIG. 10). Briefly,copy number of the region upstream of the HBG1 promoter (between the 5.2kb Fwd and Rev primers shown in FIG. 10) was defined in relation to copynumber at the RPPH1 locus as described in the methods. Allelefrequencies for the localized smaller indels are not relative to thetotal indel frequency, because the amplification of the HBG1 or HBG2site for NGS would not occur in alleles containing the 4.9 kb inversionor deletion. For example, for gRNA GCR-0001 the frequency of the 4.9 kbdeletion is 35.2%, with the remaining 64.8% (100%−35.2%, ignoringinversions) being a mixture of wildtype and smaller localized indels.Therefore the 9% single base pair A base deletion would be 5.8% of thetotal indel frequency i.e. 9% of 64.8%. Allele frequencies shown inTable 7-2 vary slightly between experiments and should not be consideredas absolute values.

TABLE 7-2 Top on-target editing repair pattern (collectively for anygRNA molecule, also referred to herein as “indel pattern”) variantsgenerated by editing cells from the first donor with gRNAs GCR-0001,GCR-0008, GCR-0010, GCR-0048, GCR-0051 and GCR-0067. Variant size,variant type (Ins = insertion, Del = deletion), reference allele,variant allele, variant start and end position relative to chromosome 11reference genome build hg38, and allele frequency are shown. The HBG2locus edited with gRNA GCR-0001 shows no significant localized indels,but based on the qPCR results it does result in a 4.9kb deletion. gRNASize Variant Variant start and Allele name (bp) Type Reference alleleallele end position frequency GCR- 4928 Del Not shown Not5250172-5255100 35.2% 0001 shown (HBG1) −1 Del CA C 5250171-5250172 9.0%1 Ins A AT 5250172-5250173 8.1% −1 Del AT A 5250172-5250173 8.0% −2 DelATA A 5250172-5250174 5.1% 1 Ins A AA 5250172-5250172 2.6% GCR- 4928 DelNot shown Not 5250150-5255078 40.4% 0008 shown (HBG1) 1 Ins T TA5250150-5250151 50.5% −2 Del AGC A 5250151-5250153 3.4% −1 Del TA T5250150-5250151 3.3% −1 Del AG A 5250151-5250152 2.4% −4 Del TAGCT T5250150-5250154 2.2% GCR- 4928 Del Not shown Not 5250150-5255078 40.4%0008* shown (HBG2) 1 Ins T TA 5255078-5255079 55.8% −19 DelCCCTTTAGCTAGTTTTCT A 5255073-5255092 5.6% TC (SEQ ID NO: 266) −2 Del AGCA 5255079-5255081 2.9% −6 Del TTTAGCT T 5255076-5255082 2.8% −1 Del TA T5255078-5255079 2.6% GCR- 4928 Del Not shown Not 5250151-5255079 43.6%0010 shown (HBG1) 1 Ins A AG 5250151-5250152 5.6% −4 Del AGCTA A5250151-5250155 1.8% 1 Ins A AA 5250151-5250152 1.6% GCR- 4928 Del Notshown Not 5250151-5255079 43.6% 0010* shown (HBG2) 1 Ins A AG5255079-5255080 5.7% −19 Del CCCTTTAGCTAGTTTTCT A 5255073-5255092 2.6%TC (SEQ ID NO: 266) −52 Del GCCTTGTT.....TTCCCTTTA G 5255027-52550792.0% −4 Del TAGCT T 5255078-5255082 1.5% −6 Del TTTAGCT T5255076-5255082 1.5% GCR- 4928 Del Not shown Not 5250187-5255115 43.5%0048 shown (HBG1) −1 Del AC A 5250187-5250188 40.0% −2 Del ACT A5250187-5250189 10.1% −4 Del ACTTC A 5250187-5250191 5.2% 1 Ins A AA5250187-5250188 1.9% −1 Del GA G 5250186-5250187 2.1% GCR- 4928 Del Notshown Not 5250187-5255115 43.5% 0048 shown (HBG2) −1 Del AC A5255115-5255116 42.4% −2 Del ACT A 5255115-5255117 8.9% −3 Del ACTT A5255115-5255118 2.7% −4 Del ACTTC A 5255115-5255119 2.5% −15 DelGGACTTCTTTTGTCAG G 5255113-5255128 2.5% (SEQ ID NO: 267) GCR- 4928 DelNot shown Not 5250150-5255078 44.9% 0051* shown (HBG1) 1 Ins T TA5250150-5250151 39.4% −1 Del TA A 5250150-5250151 1.3% −2 Del AGC A5250151-5250153 0.7% −14 Del TTAGCTAGTTTCCTT T 5250149-5250163 0.9% (SEQID NO: 268) −6 Del TTTAGCT T 5250148-5250154 0.6% GCR- 4928 Del Notshown Not 5250150-5255078 44.9% 0051 shown (HBG2) 1 Ins T TA5255078-5255079 52.5% −19 Del CCCTTTAGCTAGTTTTCT C 5255073-5255092 4.9%TC (SEQ ID NO: 269) −2 Del AGC A 5255079-5255081 3.2% −4 Del TAGCT T5255078-5255082 2.4% −1 Del TA T 5255078-5255079 2.2% GCR- 4928 Del Notshown Not 5250099-5255027 55.3% 0067 shown (HBG1) −6 Del GCCTTTG G5250093-5250099 11.3% −1 Del TG T 5250098-5250099 9.4% −1 Del GC G5250099-5250100 7.0% −5 Del GCCTTG G 5250099-5250104 5.4% −2 Del GCC G5250099-5250101 3.4% GCR- 4928 Del Not shown Not 5250099-5255027 55.3%0067 shown (HBG2) −6 Del GCCTTTG G 5255021-5255027 36.1% −5 Del GCCTTG G5255027-5255032 15.7% −1 Del TG T 5255026-5255027 4.0% −1 Del GC G5255027-5255028 2.4% −2 Del GCC G 5255027-5255029 2.1% *HBG1/2 targetsite with mismatches.

High overall editing frequencies at the gamma globin locus, includinghigh frequencies of large 4.9 kb deletions between the HBG1 and HBG2promotor targeting sites, were observed after electroporating cells withRNPs containing sgRNA of the indicated targeting domain. The on-targetediting patterns shown were identified in cells which generatedincreased F cells after erythroid differentiation, as described. Inembodiments, the indel pattern for any gRNA molecule described hereinincludes the most frequent 1, 2, 3, 4, 5 or 6 indels detected at theHBG1 locus. In embodiments, the indel pattern for any gRNA moleculedescribed herein includes the most frequent 1, 2, 3, 4, 5 or 6 indelsdetected at the HBG2 locus. In embodiments, the indel pattern for anygRNA molecule described herein includes the most frequent 1, 2, 3, 4, 5or 6 indels detected at the HBG1 locus and the most frequent 1, 2, 3, 4,5 or 6 indels detected at the HBG2 locus, e.g., as described in Table7-2 (but not double-counting the approximately 4.9 kb deletion).

Example 2.4: Exemplary Editing Patterns in Isolated Sub-Populations ofHSPCs after Gamma Globin Promoter Region Editing

Methods:

Methods are as in Example 2.1, with the following modifications.

Human CD34⁺ cell culture. Human CD34+ cells were isolated from G-CSFmobilized peripheral blood from adult donors (Hemacare catalog#M001F-GCSF-3) using immunoselection (Miltenyi) according to themanufacturer's instructions and expanded for 2 days prior toelectroporation with RNP complexes.

Assembly of Cas9 and guide RNA ribonucleoprotein (RNP) complexes,preparation of HSPC, and electroporation of RNP into HSPC. For formationof RNP using single guide RNAs (sgRNAs), 12 μg of each of sgRNA, 12 μgof CAS9 protein and 1 μL of 10×CCE buffer (20 mM HEPES, 100 mM KCL, 5 mMMgCL2, 5% Glycerol and freshly added 1 mM DTT) were combined in a totalvolume of 10 ul, then incubated at 37° C. for 5 min. Cell density in P3buffer + supplement was 1.3×10⁸/mL. Seven replicate electroporation wereperformed using GCR-0067, and cells were combined post-electroporation.For the None/control condition, cells were transferred directly from P3buffer into expansion medium without electroporation or addition ofCas9. Following electroporation with RNP complexes, cells were expandedfor an additional 3 days prior to flow cytometry cell sorting.

Flow cytometry cell sorting for analysis of editing efficiency inhematopoietic stem and progenitor subpopulations. Edited cell culturesat 3 days post-electroporation were harvested and incubated withanti-CD34 (BD Biosciences, Cat #348057), anti-CD38 (BD Biosciences, Cat#560677), anti-CD90 (BD Biosciences, Cat #559869), anti-CD45RA (BDBiosciences, Cat #563963), anti-CD49f (BD Biosciences, Cat #562598) inFACS staining buffer consisting of HBSS (GE Life Sciences, Cat.#SH30588.01) supplemented with 2% FBS (Omega Scientific, Cat. #FB-11)and 2 mM EDTA (Corning Cat. #46-034-CL). Cells were washed with FACSstaining buffer, and cell viability was determined by addition of DAPI(4′,6-Diamidino-2-Phenylindole). Multicolor FACS analysis was performedon a FACS Aria cell sorter (BD Biosciences). Discrimination betweennegative and positive cell populations was determined by gates set usingcontrol stained cell cultures, in which each antibody was individuallyreplaced with an isotype and fluorochrome-conjugate matched non-specificcontrol antibody (BD Biosciences Cat #550854, 554680, 563437, 557872,and 562602). Purity of sorted cells was confirmed by post-sort puritycheck.

Genomic DNA preparation and next generation sequencing (NGS). GenomicDNA was prepared from unedited HSPC and sorted subpopulations of editedHSPC at 3 days post-electroporation using the DNeasy Blood & Tissue Kit(Qiagen Cat #69504). NGS analysis described below showed no significantediting in control unedited samples.

NGS library preparation and sequencing of amplicons was carried out asdescribed in Example 2.1. NGS sequencing data QC and variant analysiswas performed as described in Example 2.1.

Quantification of large deletions. Large deletions between targetingsites at the HBG1 and HBG2 promotors were quantified by digital dropletPCR (ddPCR) in non-competitive assay format for copy numberdetermination according to manufacturer's recommendations. Briefly, gDNAwas combined with ddPCR SuperMix for Probes (no dUTPs) (BioRad Cat#1863024), HindIII-HF restriction enzyme (NEB Cat #R3104S) and eachprimer probe mix, transferred to a DG8 cartridge along with DropletGeneration Oil for Probes (BioRad Cat #1863005) and droplets generatedwith a QX200 droplet generator (BioRad). Droplets were subject to PCR ona C1000 Touch Thermal Cycler with 96-Deep Well Reaction Module (BioRad),followed by detection with QX200 droplet reader (BioRad). Copies per ulwas determined by analysis with QuantaSoft software (BioRad). A customprimer probe set (Life Technologies Cat #APZW76R, PN4331348, Forwardprimer: ACGGATAAGTAGATATTGAGGTAAGC (SEQ ID NO: 270), Reverse primer:GTCTCTTTCAGTTAGCAGTGG (SEQ ID NO: 271), FAM TaqMan Probe:ACTGCGCTGAAACTGTGGCTTTATAG (SEQ ID NO: 272)) used to amplify gDNA withinthe HBG1-HBG2 intergenic region. A TaqMan Copy Number Reference Assay,human, RNase P (Thermo Fisher cat #4403326) was used as a referenceamplicon. Copies per ul for HBG1-HBG2 and RNase P amplicons were withinthe manufacturer's reported linear range. Percent deletion was reportedas 100% times 1 minus the ratio of copies per ul for HBG1-HBG2 and RNaseP amplicons. Unedited control samples had calculated percent deletionsup to 13%, which may reflect background in the assay.

Results:

Without being bound by theory, the CD34+ HSPC population is thought tocontain cells of various potential for engraftment, self-renewal andcell fate, with additional markers further enriching in cells withshared properties, including engrafting long-term hematopoietic stemcells (Notta, Science, 2011 Jul. 8; 333(6039):218-21. doi:10.1126/science.1201219; Huntsman, Blood. 2015 Sep. 24; 126(13):1631-3.doi: 10.1182/blood-2015-07-660670; each incorporated herein by referencein their entirety). Without being bound by theory, such subpopulationsmay be of particular therapeutic benefit in gene edited cell transplant,as they may reconstitute the hematopoietic system at different timespost-transplant. Thus, cells were subjected to flow cytometry tocharacterize editing efficiency in HSPC subpopulations. Five cellpopulations were isolated, first CD34+ cells, followed by a 4-way sortfor CD34+CD45RA−CD38+, CD34+CD45RA−CD38−CD90−CD49f+,CD34+CD45RA−CD38−CD90+CD49f+ and CD34+CD45RA−CD38−CD90−CD49f− cellsusing the strategy shown (FIG. 11).

Each of these sorted populations was analyzed for editing byindividually sequencing the HBG1 and HBG2 target site localized regions,as well as by detection of deletion of the intervening HBG1-HBG2 region.HBG1-HBG2 deletion (i.e., excision) frequencies were consistently highacross subpopulations, ranging from 69 to 81 percent (FIG. 12).Localized amplification at the HBG1 and HBG2 promotor, individually, andNGS was used to determine % editing at each site (small indels). Alleleswith inversions or deletions caused by targeting at both promotors wouldnot be amplified in the assay. Of the alleles without HBG1-HBG2 deletionor inversion, editing was similarly high across sub-populations, rangingfrom 53 to 73 percent for HBG1 and 75 to 91 percent for HBG2 Minimaltotal editing was estimated by combining the percentage of HBG1-HBG2deleted genomes with the percentage of non-HBG1-HBG2 deleted genomeswith editing at HBG2 (minimum total editing=deletion plus % undeleted[100 minus % deletion, ignoring inversions] times the % edited at HBG2).Localized small indels at HBG1 were not included, because we could notdetermine what proportion of % editing at HBG1 was an allele with indelslocalized to HBG1 only and what proportion was an allele withco-existing indels at both HBG1 and HBG2 Minimal total editing wasestimated to range from 92 to 97 percent across sub-populations. Editingpatterns for indels localized to the HBG1 and

HBG2 target sites were also similar across sub-populations, with themost frequently observed editing patterns common across sub-populations.For all sub-populations and each site, HBG1 or HBG2, the top three indelpatterns included a 6 bp deletion, a 5 bp deletion and a 1 bp deletion(FIG. 13A (HBG1 locus); FIG. 13B (HBG2 locus)). In summary, theconsistent editing frequencies and patterns between total HSPC andsub-fractions, including defined CD34+CD45RA−CD38−CD90+CD49f+ long-termhematopoietic stem cells, supports the utility of the herein describedgene editing approach for HSPC transplant.

Example 2.5: Colony Forming Ability of HSPCs after Gamma Globin PromoterRegion Editing

Methods:

Methods are as in Example 2.1, with the following modifications.

Human CD34⁺ cell culture. Human CD34+ cells were derived from bonemarrow from adult healthy donors (Lonza catalog #2M-101D). Cells werethawed then expanded for 2 days prior to electroporation with RNPcomplexes.

Assembly of Cas9 and guide RNA ribonucleoprotein (RNP) complexes,preparation of HSPC, and electroporation of RNP into HSPC. For formationof RNP using single guide RNAs (sgRNAs), 12 μg of each of sgRNA, 12 μgof CAS9 protein and 1 μL of 10×CCE buffer (20 mM HEPES, 100 mM KCL, 5 mMMgCL2, 5% Glycerol and freshly added 1 mM DTT) were combined in a totalvolume of 10 ul, then incubated at 37° C. for 5 min. Cell density in P3buffer + supplement was 6.64×10⁶/mL. Two electroporation replicates wereperformed with cells from each of two independent donors. For theNone/control condition, vehicle rather than sgRNA was added. Followingelectroporation with RNP complexes, cells were returned to expansionmedium.

Genomic DNA preparation and next generation sequencing (NGS). GenomicDNA was prepared from unedited HSPC and sorted subpopulations of editedHSPC at 3 days post-electroporation using the DNeasy Blood & Tissue Kit(Qiagen Cat #69504). NGS analysis described below showed no significantediting in control samples electroporated with Cas9 alone.

NGS library preparation and sequencing of amplicons was carried out asdescribed in Example 2.1. NGS sequencing data QC and variant analysiswas performed as described in Example 2.1.

Quantification of large deletions. Large deletions between targetingsites at the HBG1 and HBG2 promotors were quantified by digital dropletPCR (ddPCR) in non-competitive assay format for copy numberdetermination according to manufacturer's recommendations. Briefly, gDNAwas combined with ddPCR SuperMix for Probes (no dUTPs) (BioRad Cat#1863024), HindIII-HF restriction enzyme (NEB Cat #R3104S) and eachprimer probe mix, transferred to a DG8 cartridge along with DropletGeneration Oil for Probes (BioRad Cat #1863005) and droplets generatedwith a QX200 droplet generator (BioRad). Droplets were subject to PCR ona C1000 Touch Thermal Cycler with 96-Deep Well Reaction Module (BioRad),followed by detection with QX200 droplet reader (BioRad). Copies per ulwas determined by analysis with QuantaSoft software (BioRad). A customprimer probe set (Life Technologies Cat #APZW76R, PN4331348, Forwardprimer: ACGGATAAGTAGATATTGAGGTAAGC (SEQ ID NO: 273), Reverse primer:GTCTCTTTCAGTTAGCAGTGG (SEQ ID NO: 274), FAM TaqMan Probe:ACTGCGCTGAAACTGTGGCTTTATAG (SEQ ID NO: 275)) used to amplify gDNA withinthe HBG1-HBG2 intergenic region. A TaqMan Copy Number Reference Assay,human, RNase P (Thermo Fisher cat #4403326) was used as a referenceamplicon. Copies per ul for HBG1-HBG2 and RNase P amplicons were withinthe manufacturer's reported linear range. Percent deletion was reportedas 100% times 1 minus the ratio of copies per ul for HBG1-HBG2 and RNaseP amplicons. Unedited control samples had calculated percent deletionsup to 9%, which may reflect background in the assay.

Colony forming unit cell assay. Two days following RNP delivery, viablecells were enumerated by flow cytometry on an LSRFortessa (BDBiosciences) using TruCount tubes (BD Biosciences Cat #340334) accordingto the manufacturer's recommendations. Inviable cells were discriminatedusing DAPI (4′,6-Diamidino-2-Phenylindole). Analysis was performed usingFlowJo software (Tree Star). For the colony forming unit (CFU) assay,cells and 1× antibiotic/antimycotic (Gibco, Cat. #10378-016) were addedto MethoCult H4034 Optimum (Stemcell Technologies) methylcellulosemedium (StemCell Technologies) and 1 mL was plated in triplicate inSmartDish plates (StemCell Technologies). The culture dishes wereincubated in a humidified incubator at 37° C. Cultures were imaged onday 14 post-plating using a StemVision (Stemcell Technologies). Colonieswere manually scored using Colony Marker software (StemcellTechnologies). Colony number per well (average of three wells) wasdivided by the number of cells plated per ml of Methocult (ranged from226 to 318) and multiplied by 1000 to obtain the CFU frequency per 1000cells.

Results:

We have shown here that HSPCs function in colony formation assayfollowing the targeted disruption of specific sequences within the HBG1and HBG2 promoter regions associated with the production of F-cells. Thegenome edited and unedited HSPC were evaluated for progenitor cellcomposition and differentiation potential using a colony forming unitassay. Colonies were counted and classified as deriving from erythroidprogenitor cells (CFU-erythroid [CFU-E] and burst-forming unit-erythroid[BFU-E]), granulocyte and/or macrophage progenitor cells(CFU-granulocyte, macrophage [CFU-GM]; CFU-granulocyte [CFU-G]; andCFU-macrophage [CFU-M]), or multi-potential progenitor cells(CFU-granulocyte, erythrocyte, macrophage, megakaryocyte [CFU-GEMM]).

TABLE 8 List of select gRNAs targeting the HBG1 and HBG2 promoter regionused in the current study to edit cells from the first independentdonor. BFU- CFU- total % % E/CFU- CFU- GEMM colonies Guide RNA % % HBG1-approx. E per G/M/GM per per targeting edited edited HBG2 min. 1000 per1000 1000 1000 domain ID HBG1 HBG2 deletion editing cells cells cellscells None/control n/a n/a 8.6 n/a 48 162 3 213 GCR-0008 80.7 78.9 42.688.9 18 134 2 154 GCR-0010 34.9 18.0 29.5 54.1 25 118 4 148 GCR-004881.4 84.0 40.6 90.5 28 130 0 158 GCR-0051 51.0 74.9 41.2 85.2 28 133 2164 GCR-0067 82.9 88.6 51.3 94.4 25 123 5 153

TABLE 9 List of select gRNAs targeting the HBG1 and HBG2 promoter regionused in the current study to edit cells from the second independentdonor. BFU- CFU- total % % E/CFU- CFU- GEMM colonies Guide RNA % % HBG1-approx. E per G/M/GM per per targeting edited edited HBG2 min. 1000 per1000 1000 1000 domain ID HBG1 HBG2 deletion editing cells cells cellscells None/control n/a n/a 8.9 n/a 90 241 12 343 GCR-0008 82.3 85.2 47.092.1 51 151 4 206 GCR-0010 42.3 35.1 43.0 67.1 56 162 5 223 GCR-004868.7 76.1 41.7 86.0 49 129 7 186 GCR-0051 55.2 82.7 41.3 89.9 32 157 6196 GCR-0067 81.2 88.9 47.1 94.1 39 169 9 217

Both donors electroporated with RNPs with the indicated gRNAs hadefficient editing, both indels localized to the HBG1 and HBG2 promotortarget/off-target sites, as well as deletions of the intervening region(Tables 8 and 9). Edited cultures were associated with a drop in overallcolony forming capacity (Tables 8 and 9), possibly indicating decreasedfitness of cells undergoing editing. Despite this reduction in totalcolony number, erythroid, granulocyte/macrophage, and multi-potentialcolonies were all observed in at least one donor (Tables 8 and 9).Furthermore, there were minimal differences in the proportion of colonytypes between unedited and edited samples (FIG. 14), indicating thatcell cultures edited at these target sites did not have skeweddifferentiation capacity.

Example 2.6: Cell Proliferation of Hspcs In Vitro after Gamma GlobinPromoter Region Editing

Methods:

Methods are as in Example 2.1, with the following modifications.

Human CD34⁺ cell culture. Human CD34+ cells were derived from bonemarrow from adult healthy donors (Lonza catalog #2M-101D and Hemacarecatalog #BM34-C). Cells were thawed then expanded for 2 days prior toelectroporation with RNP complexes.

Assembly of Cas9 and guide RNA ribonucleoprotein (RNP) complexes,preparation of HSPC, and electroporation of RNP into HSPC. For formationof RNP using single guide RNAs (sgRNAs), 12 μg of each of sgRNA, 12 μgof CAS9 protein and 1 μL of 10×CCE buffer (20 mM HEPES, 100 mM KCL, 5 mMMgCL2, 5% Glycerol and freshly added 1 mM DTT) were combined in a totalvolume of 10 ul, then incubated at 37° C. for 5 min. Cell density in P3buffer + supplement was 1×10⁷ to 2.5×10⁷/mL. One electroporationreplicate was performed with cells from each of three independentdonors. For the None/control condition, vehicle rather than sgRNA wasadded. Following electroporation with RNP complexes, cells were returnedto expansion medium.

Genomic DNA preparation and next generation sequencing (NGS). GenomicDNA was prepared from unedited and edited HSPC at 3 dayspost-electroporation using Quick Extract DNA Extraction Solution(Epicentre Cat #QE09050) or the DNeasy Blood & Tissue Kit (Qiagen Cat#69504)NGS analysis described below showed no significant editing incontrol samples electroporated with Cas9 alone.

NGS library preparation and sequencing of amplicons was carried out asdescribed in Example 2.1. NGS sequencing data QC and variant analysiswas performed as described in Example 2.1.

Cell proliferation and phenotyping. Two days following RNP delivery,viable cells were enumerated by flow cytometry on an LSRFortessa (BDBiosciences) using TruCount tubes (BD Biosciences Cat #340334) accordingto the manufacturer's recommendations. Inviable cells were discriminatedusing DAPI (4′,6-Diamidino-2-Phenylindole), and CD34+ and CD34+CD90+cell content was determined by inclusion of anti-CD34 (BD BiosciencesCat #348057, BD Biosciences Cat #340666 or eBioscience Cat #25-0349-425)and anti-CD90 (BD Biosciences Cat #559869) in FACS staining bufferconsisting of HBSS (GE Life Sciences Cat. #SH30588.01) supplemented with2% FBS (Omega Scientific Cat. #FB-11) and 2 mM EDTA (Corning Cat.#46-034-CL). Analysis was performed using FlowJo software (Tree Star).Cells were then seeded into expansion medium at either 2.0×10⁴ or1.0×10⁵ viable cells/ml and cultured for 7 days. For cultures seeded at1.0×10⁵/ml, fresh medium was added during the culture period for a 3 or4-fold dilution. After 7 days culture, the cells were once againenumerated as above. After 7 days culture, cells were additionallyanalyzed for surface marker expression after staining with the followingantibody panels. Panel 1: Antibodies specific for CD38 (FITC-conjugate,BD Biosciences #340926, clone HB7), CD133 epitope 1 (PE-conjugate,Miltenyi #130-080-801, clone AC133), CD34 (PerCP-conjugate, BDBiosciences #340666, clone 8G12), CD90 (APC-conjugate, BD Biosciences#559869, clone 5E10), CD45RA (Pe-Cy7-conjugate, eBioscience #25-0458-42,clone HI100). Panel 2: CD34 (PerCP-conjugate, BD Biosciences #340666,clone 8G12), CD33 (PE-Cy7-conjugate, BD Biosciences #333946, cloneP67.6), CD14 (APC-H7-conjugate, BD Biosciences #560270, clone MφP9),CD15 (PE-conjugate, Biolegend #301905, clone HI98). Panel 3: CD34(PerCP-conjugate, BD Biosciences #340666, clone 8G12), CD41a(APC-H7-conjugate, BD Biosciences #561422, clone HIPS), CD71(FITC-conjugate, BD Biosciences #555536, clone M-A712), CD19(PE-conjugate, BD Biosciences #340720, clone SJ25C1), CD56(APC-conjugate, Biolegend #318310, clone HCD56). Corresponding isotypecontrol antibody panels were used to stain cultures in parallel.Inviable cells were discriminated by DAPI(4′,6-Diamidino-2-Phenylindole) staining Stained samples were analyzedon an LSRFortessa flow cytometer (BD Biosciences) for cell surfaceprotein expression. The results were analyzed using Flowjo, and datawere presented as % of the DAPI negative viable cell population.

Results:

We have shown here that HSPCs can expand with typical cellularcomposition in vitro following the targeted disruption of specificsequences within the HBG1 and HBG2 promoter regions associated with theproduction of F-cells. The genome edited and unedited HSPC wereevaluated for proliferation capacity and cell composition in cultureconditions that promote expansion of HSPC. Replicate independent donorselectroporated with RNPs containing GCR-0067 had efficient editing(Table 9-2).

TABLE 9-2 Percent edited amplicons of HBG1 or HBG2 promotor region incells from three independent donors using indicated gRNA. Guide RNA % %% % % % targeting edited edited edited edited edited edited domain HBG1,HBG2, HBG1, HBG2, HBG1, HBG2, ID donor A donor A donor B donor B donor Cdonor C GCR- 62.7 64.5 57.0 73.4 53.1 56.9 0067

Edited cultures were associated with a drop in overall proliferationcapacity, possibly indicating mildly decreased fitness of cellsundergoing editing, although this did not reach significance over threeindependent cell donors (FIG. 15) Similar reductions were observedwithin the hematopoietic stem cell enriched CD34+CD90+ population as inthe total CD34+ population, indicating that this population is notdifferentially effected (FIG. 15). Under these culture conditions, boththe HSPC and differentiated progeny are expected to be present, thus,cellular composition was further analyzed for expression of a morecomprehensive panel of cell surface markers. Discrimination of thesecell populations is exemplified in FIGS. 16A-16B. Cellular compositionwas similar between genome edited and unedited cultures across threeindependent cell donors, with no significant difference in a givenpopulation by unpaired t-test (FIG. 17). The large error bars for theCD33+ population result from a single donor with negligible CD33+ cellsin both the genome edited and unedited cultures (FIG. 17).

Example 3: Evaluation of Cas9 Variants

Evaluation in CD34+ Hematopoietic Stem Cells

We evaluated 14 purified Streptococcus pyogenes Cas9 (SPyCas9) proteinsby measuring their efficiency of knocking out the beta-2-microglobulin(B2M) gene in primary human hematopoietic stem cells (HSCs). Theseproteins were divided into 3 groups: the first group consisted ofSPyCas9 variants with improved selectivity (Slaymaker et al. 2015,Science 351: 84 (e1.0, e1.1 and K855A); Kleinstiver et al. 2016, Nature529: 490 (HF)). The second group consisted of wild type SPyCas9 withdifferent numbers and/or positions of the SV40 nuclear localizationsignal (NLS) and the 6× Histidine (His6) (SEQ ID NO: 247) or 8×Histidine (His8) tag (SEQ ID NO: 248) with or without a cleavable TEVsite, and a SPyCas9 protein with two cysteine substitutions (C80L,C574E), which have been reported to stabilize Cas9 for structuralstudies (Nishimasu et al. 2014, Cell 156:935). The third group consistedof the same recombinant SPyCas9 produced by different processes (FIG.6). B2M knockout was determined by FACS and next generation sequencing(NGS).

Methods

Materials

-   1. Neon electroporation instrument (Invitrogen, MPK5000)-   2. Neon electroporation kit (Invitrogen, MPK1025)-   3. crRNA (targeting domain sequence of GGCCACGGAGCGAGACAUCU (SEQ ID    NO: 276), complementary to a sequence in the B2M gene, fused to SEQ    ID NO: 201)-   4. tracrRNA (SEQ ID NO: 224)-   5. Cas9 storage buffer: 20 mM Tris-C1, pH 8.0, 200 mM KCl, 10 mM    MgCl₂-   6. Bone marrow derived CD34+ HSCs (Lonza, 2M-101C)-   7. Cell culture media (Stemcell Technologies, StemSpam SFEM II with    StemSpam CC-100)-   8. FACS wash buffer: 2% FCS in PBS-   9. FACS block buffer: per mL PBS, add 0.5 ug mouse IgG, 150 ug Fc    block, 20 uL FCS-   10. Chelex suspension: 10% Chelex 100 (bioRad, Cat #142-1253) in H₂O-   11. Anti-B2M antibody: Biolegend, cat #316304

Process

Thaw and grow the cells following Lonza's recommendations, add mediaevery 2-3 days. On day 5, pellet the cells at 200×g for 15 min, washonce with PBS, resuspend the cells with T-buffer from NEON kit at2×10⁴/uL, put on ice. Dilute Cas 9 protein with Cas9 storage buffer to 5mg/ml. Reconstitute crRNA and tracrRNA to 100 uM with H₂O. Theribonucleoprotein (RNP) complex is made by mixing 0.8 uL each of CAS 9protein, crRNA and tracrRNA with 0.6 uL of Cas9 storage buffer, incubateat room temperature for 10 min. Mix 7 uL of HSCs with RNP complex fortwo minutes and transfer the entire 10 uL into a Neon pipette tip,electroporate at 1700 v, 20 ms and 1 pulse. After electroporation,immediately transfer cells into a well of 24-well plate containing 1 mlmedia pre-calibrated at 37° C., 5% CO₂. Harvest cells 72 hrspost-electroproation for FACS and NGS analysis.

FACS: take 250 uL of the cells from each well of 24-well plate, to wellsof 96-well U-bottom plate and pellet the cells. Wash once with 2% FCS(fetal calf serum)-PBS. Add 50 uL FACS block buffer to the cells andincubate on ice for 10 minutes, add 1 uL FITC labeled B2M antibody andincubate for 30 minutes. Wash with 150 uL FACS wash buffer once followedby once more with 200 uL FACS wash buffer once. Cells were resuspendedin 200 uL FACS buffer FACS analysis.

NGS sample prep: transfer 250 uL of cell suspension from each well ofthe 24-well plate to a 1.5 ml Eppendorf tube, add 1 mL PBS and pelletthe cells. Add 100 uL of Chelex suspension, incubate at 99° C. for 8minutes and vortex 10 seconds followed by incubating at 99° C. for 8minutes, vortex 10 seconds. Pellet down the resin by centrifuging at10,000×g for 3 minutes and the supernatant lysate is used for PCR. Take4 uL lysate and do PCR reaction with the b2m primers (b2mg67F:CAGACAGCAAACTCACCCAGT (SEQ ID NO: 277), b2mg67R: CTGACGCTTATCGACGCCCT(SEQ ID NO: 278)) using Titanium kit (Clonetech, cat #639208) and followthe manufacturer's instruction. The following PCR conditions are used: 5minutes at 98° C. for 1 cycle; 15 seconds at 95° C., 15 seconds at 62°C., and 1 minute at 72° C. for 30 cycles; and finally 3 minutes at 72°C. for 1 cycle. The PCR product was used for NGS.

NGS library preparation and sequencing of amplicons was carried out asdescribed in Example 2.1. NGS sequencing data QC and variant analysiswas performed as described in Example 2.1.

Statistics: The percentage of B2M KO cells by FACS and the percentage ofindels by NGS are used to evaluate the CAS 9 cleavage efficiency. Theexperiment was designed with Cas9 as fixed effect. Each experiment isnested within donors, as nested random effects. Therefore, the mixedlinear model was applied for the analysis of FACS and NGS data.

Results

In order to normalize the experimental and donor variations, we graphedthe relative activity of each protein to iProt105026, the originaldesign with two SV40 NLS flanking the wild type SPyCas9 and the His6 tag(SEQ ID NO: 247) at the C-terminal of the protein (FIG. 6). Thestatistical analysis shows that compared with the reference Cas9 proteiniProt105026, iProt106331, iProt106518, iProt106520 and iProt106521 arenot significantly different in knocking out B2M in HSCs, while the othervariants tested (PID426303, iProt106519, iProt106522, iProt106545,iProt106658, iProt106745, iProt106746, iProt106747, iProt106884) arehighly significantly different from the reference iProt105026 inknocking out B2M in HSCs. We found that moving the His6 tag (SEQ ID NO:247) from the C-terminal to N-terminal (iProt106520) did not affect theactivity of the protein (FIG. 6). One NLS was sufficient to maintainactivity only when it was placed at the C-terminal of the protein(iProt106521 vs. iProt106522, FIG. 6). Proteins purified from process 1had consistent higher knockout efficiency than those from processes 2and 3 (iProt106331 vs. iProt106545 & PID426303, FIG. 6). In general, theSPyCas9 variants with a reported improved selectivity were not as activeas the wild type SPyCas9 (iProt106745, iProt106746 and iProt106747, FIG.6). Interestingly iProt106884 did not cut the targeting site. This isconsistent with the report by Kleinstiver et al that this variant failedto cut up to 20% of the legitimate targeting sites in mammalian cells(Kleinstiver et al. 2016, Nature 529: 490). Finally, the Cas9 variantwith two cysteine substitutions (iProt106518) maintained high levels ofenzymatic activity (FIG. 6).

Example 4. Quantification of Hemoglobin Subunit Changes UponGene-Editing by Capillary Electrophoresis Mass Spectrometry

Upon gene-editing of HSCs, in addition to capturing fetal hemoglobinproduction by flow cytometry, changes in individual hemoglobin subunitsin erythroid cells were also measured by capillary electrophoresis massspectrometry (CE-MS). CD34+ cells derived from the peripheral blood ofsickle cell patients were either mock edited in the presence of Cas9protein (with two NLS and one His Tag, iProt106331) and no guide RNA, orgene-edited in the presence of both Cas9 protein and guide RNA sg0067,and differentiated into erythroid lineage in culture as describedpreviously. At day 14 of erythroid differentiation, same number of cellsfrom each condition was stained with FITC-conjugated antibodyrecognizing fetal hemoglobin (HbF), and cells were subjected to flowcytometry to quantify HbF induction upon gene editing. Flow cytometryreported 30-50% upregulation of HbF+ cells (Table 10). In parallel,varying amounts of erythroid cells (6, 12, 66, and 99 million) percondition were harvested and subjected to CE-MS to quantifyalpha-globin, normal beta-globin (HbB), sickle beta-globin (HbS), andfetal gamma-globin (HbF) subunits and data were normalized with theamount of input cells. CE-MS was able to detect all globin subunits inas few as 600 cells/ul concentration. Results showed that edited sicklecell patient samples had a ˜40% increase in gamma-globin and aconcurrent 50% decrease in sickle beta-globin (FIG. 18).

TABLE 10 Description of treatment and cell quantity of samples subjectedto CE-MS quantification of globin subunits. Fetal hemoglobin expressionof these samples was also measured by flow cytometry using anti-HbFantibody conjugated to a fluorophore. HbF + Cells Measured Number byFlow of Cells Cytometry Donor ID Sample ID Sample Description (1 × 10e6)(%) Sickle cell 1 Mock edited, no sgRNA, 99.2 55.5 patient 1 erythroiddifferentiated 2 Gene edited with sg0067, 66.6 81.15 erythroiddifferentiated Sickle cell 3 Mock edited, no sgRNA, 12.6 59.70 patient 2erythroid differentiated 4 Gene edited with sg0067, 5.5 81.85 erythroiddifferentiated

Example 5. Gene-Edited Hematopoietic Stem Cells (HSCs) were Capable ofLong-Term Engraftment and Support Multi-Lineage Reconstitution

To evaluate the stem cell function of gene-edited HSCs, edited humanHSCs were transplanted into immunocompromised mice to examine long-termhematopoietic regeneration. Five hundred thousand human bonemarrow-derived CD34+ cells were thawed at day 0, electroporated at day 3with either a mock RNP complex (Cas9 alone and no gRNA) or RNP complexformed with Cas9 and sgRNA comprising the targeting domain of CR001128(sometimes referred to herein at sg1128)(AUCAGAGGCCAAACCCUUCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 181), comprisingthe targeting domain sequence of AUCAGAGGCCAAACCCUUCC (SEQ ID NO: 180)).Cell were subsequently maintained in the same concentration and mediumdescribed in Example 2.1, and at day 6 of culture, all cells from eachtreatment was transplanted into 2 Gray sublethally irradiatedNOD.Cg-Prkdc^(scid)Il2^(tm1Wjl)/SzJ (NSG) mice (FIG. 19). We observedapproximately 30-40% human cell engraftment in the bone marrow of therecipients at 16 weeks post-transplant. Furthermore, this engraftmentlevel was comparable to mock treatment control, suggesting thatgene-editing does not impair the long-term engraftment ability of HSCs(FIG. 20). Gene-edited, transplanted HSCs sustained normal myeloid, Blymphoid, and T lymphoid cell regeneration at 4, 8, 12, 16 weekspost-transplant (FIG. 21). This data demonstrate that gene-edited HSCswere capable of long-term engraftment and support normal multi-lineagereconstitution including out to 16 weeks post transplantation.

Example 6. Gene-Edited, Long-Term Engrafted HSCs were Capable ofSustained Fetal Hemoglobin (HbF) Production

In a separate study, we evaluated the stem cell function of HSCsgene-edited with gRNAs from the gamma globin promoter region (sg-G0008,sg-G0051, sg-G0010, sg-G0048, sg-G0067 in Table 11) in comparison togRNA from the erythroid-specific enhancer region of the BCL11A gene(sg-G1128). Human HSCs edited with these gRNAs were transplanted intoimmunocompromised NSG mice to examine hematopoietic regeneration (FIG.22).

TABLE 11 Guide RNAs used in the transplant study, designed to target thegamma globin promoter region. Target sgRNA domain gRNA target name IDcrRNA ID domain sequence sgRNA 100mer sequence sg-G0008 GCR- CR005821GGAGAAGGAA GGAGAAGGAAACUAGCUAAAGU 0008 ACUAGCUAAA UUUAGAGCUAGAAAUAGCAAGU(SEQ ID NO: 8) UAAAAUAAGGCUAGUCCGUUAU CAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 84) sg-G0010 GCR- CR005823 GGGAGAAGGAGGGAGAAGGAAACUAGCUAAGU 0010 AACUAGCUAA UUUAGAGCUAGAAAUAGCAAGU (SEQ IDNO: 10) UAAAAUAAGGCUAGUCCGUUAU CAACUUGAAAAAGUGGCACCGA GUCGGUGCUUUU (SEQID NO: 94) sg-G0048 GCR- CR005811 ACGGCUGACA ACGGCUGACAAAAGAAGUCCGU 0048AAAGAAGUCC UUUAGAGCUAGAAAUAGCAAGU (SEQ ID NO: 48) UAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA GUCGGUGCUUUU (SEQ ID NO: 134) sg-G0051 GCR-CR005813 GGAGAAGAAA GGAGAAGAAAACUAGCUAAAGU 0051 ACUAGCUAAAUUUAGAGCUAGAAAUAGCAAGU (SEQ ID NO: 51) UAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA GUCGGUGCUUUU (SEQ ID NO: 144) sg-G0067 GCR-CR005820 ACUGAAUCGG ACUGAAUCGGAACAAGGCAAGU 0067 AACAAGGCAAUUUAGAGCUAGAAAUAGCAAGU (SEQ ID NO: 67) UAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA GUCGGUGCUUUU (SEQ ID NO: 174)

Experimental Procedure

CD34+ Thawing and Culture

Bone marrow CD34+ cells were thawed and cultured. Each vial of 1 millionbone marrow CD34+ cells was removed from liquid nitrogen, sprayed with70% ethanol and thawed rapidly in a 37° C. water bath until a small icepellet remained. The vial was sprayed with 70% ethanol and wiped. Usinga 5 ml pipet, the contents of the vial were transferred into a 50 mlfalcon tube. The cryovial was rinsed once with 1 ml pre-warmed IMDM, 10%FBS and added dropwise to the 50 ml falcon tube; 25 ml of pre-warmedIMDM, 10% FBS was slowly added over 2 min to the cells, swirling gentlyto mix. Cells were spun at 300 g for 10 min. Twenty-eight million CD34+cells were cultured in StemSpan SFEM+100 ng/ml SCF/IL6/Flt3L/TPO+500 nMCompound 4+1× Pen/Strep at 0.2-0.5×10e6 cells/ml.

RNP Electroporation

Forty-eight hours post-thaw, cells were electroporated with 1) Cas9only; or 2) Cas9 RNP complex containing sg1128 (as described above), orone of the sgRNAs in Table 11. Briefly, the RNP was prepared using a 1:2molar ratio of Cas9 protein to sgRNA resulting in a RNP mixture with 10uM Cas9 and 20 uM sgRNA. 5 ul of 10 uM RNP was added to 1 million CD34+cells in 20 ul P3 buffer (Lonza). 22 ul of the electroporation mix wastransferred to one well of a 96 well electroporation plate andelectroporated using the Lonza Amaxa 96-well Shuttle or LonzaNucleofector System, program CA-137. Immediately after electroporation,80 ul of culture media (StemSpan SFEM+100 ng/ml SCF/IL6/Flt3L/TPO+500 nMCompound 4+1× Pen/Strep) was added to the electroporated sample.

Twenty-four hours post-electroporation, 500K starting cell equivalentswere transplanted per mouse. 30,000-100,000 cells were used forerythroid differentiation to assess HbF induction post-editing. Theremaining cells were left in culture for an additional 24 h (48 h totalpost-electroporation) for NGS analysis of editing frequency.

Transplantation

Ten NSG mice/condition were irradiated 4-24 h prior to transplant with200 Rad using the RadSource X-Ray irradiator, or 2 Gray using aCesium¹³⁸ irradiator. Five hundred thousand starting cell equivalentswere transplanted per mouse through tail vein injection. Followingtransplantation, the mice were placed on an antibiotic regiment for 4-8weeks. The mice were treated in accordance with institutes' animal careprocedures and following the approved IACUC protocol. At 4, 8, 12, 16and 20 weeks peripheral blood was collected for engraftment and lineageanalysis through tail vein nick. At 8-9 weeks and 20 weeks, bone marrowwas collected for analysis (flow cytometry, Taqman qPCR and NGS asdescribed in previous protocols, for example, Example 2.1) as well asfor sorting of hCD45+CD34+ cells for erythroid differentiation asdescribed in previous protocols. At week 20, the hCD45+CD34+ cell sortand erythroid differentiation were performed.

FIG. 22 shows the schematic diagram of the transplant study to evaluatestem cell function of HSCs edited with sgRNAs from the gamma globinpromoter region (sg-G0008, sg-G0051, sg-G0010, sg-G0048, sg-G0067) incomparison to gRNA from the erythroid-specific enhancer region of theBCL11A gene (sg-G1128; also referred to as sg1128). Five hundredthousand human CD34+ cells were thawed at day 0, electroporated witheither a mock RNP complex (Cas9 alone and no gRNA) or RNP complex formedwith Cas9 (NLS-Cas9-NLS-His6 (“His6” disclosed as SEQ ID NO: 247)) andvarious gRNAs at day 3. At day 6, all cells from each condition wereharvested and transplanted into 2 Gy sublethally irradiatedNOD.Cg-Prkdc^(scid)Il2rg^(tm1Wjl)/SzJ (NSG) mice (FIG. 22). Mice werebled at 4, 8, 12, 16, and 20 weeks post-transplant. At 8 and 20 weekspost-transplant, bone marrow cells from animals were also harvested toexamine human cell engraftment in the bone marrow.

Results

Editing HSCs were Capable of Long-Term Engraftment and SupportMulti-Lineage Differentiation in NSG Mice

Results showed that sgRNAs from the gamma globin promoter regionachieved on average 20-40% bone marrow engraftment at 8 weeks followingtransplantation, comparable to sg-G1128. While engraftment from earlytime points can be contributed by short-lived hematopoietic progenitorcells, engraftment at longer time points (20 weeks) is proof ofreconstitution by long-term HSCs. Tested sgRNAs showed 5-22% bone marrowengraftment at 20 weeks post-transplant (FIGS. 23A-F and 24A and 24B).It is important to note that we injected only 500,000 mock orgenome-edited CD34+ cells into each mouse to achieve such level ofengraftment. This engraftment level was comparable to other studiestransplanting 1 million genome-edited CD34+ cells, indicating a highlyefficient engraftment of the instant cells. In addition, we observednormal recovery of myeloid, B lymphoid, and T lymphoid cells at 4, 8,12, 16, 20 weeks following transplantation (FIGS. 23A-F and 25) whencomparing all gene-edited groups to the mock edited control. These datademonstrate that by targeting the gamma globin promoter region with agRNA which does not map to any known HPFH, the genome editing strategydoes not impact the engraftment capacity of long-term HSCs, nor does italter their multi-lineage reconstitution function when transplanted intoa new host. In contrast, we can achieve robust engraftment by injecting50% less cells compared to other reported strategies. In summary, HSCsedited by these sgRNAs are capable of long-term engraftment with robustmulti-lineage reconstitution in the hematopoietic stem cell niche tosustain long-term hematopoiesis.

High Editing Efficiency was Maintained Pre- and Post-Transplantation

We examined the editing efficiency of the tested sgRNAs by NGS asdescribed in experimental procedure. Three days after gene-editing,100,000 of the gene-edited human CD34+ cells, along with mock editedCD34+ cells were subjected to NGS analysis. The remaining cells weretransplanted into NSG recipients as previously described. At 8 weeks and20 weeks post-transplant, bone marrow cells from transplanted NSG micewere harvested, and 100,000 sorted human CD45+ cells were subjected toNGS to measure editing efficiency. Results show that sgRNA sg-G1128,sg-G0048, and sg-G0067 achieved 80-90% editing, while sg-G0010demonstrated ˜45% editing (FIG. 26). When comparing editing efficiencypre-transplant to after 8 or 20 weeks of transplantation, results showedthat the edited events in the hematopoietic stem and progenitorpopulations were maintained long-term throughout the transplant period.This data demonstrate the durability of edited cells in transplantedindividual. We even observe slightly increased editing efficiency forsg-G1128 at 20 weeks post-transplant, implying that edited cells wereselected by the bone marrow microenvironment or have a survivaladvantage upon transplantation.

In another independent transplant repeat, NGS analysis revealed a rangeof editing achieved with the gRNAs tested, with sg-G1128 resulting ingreater than 90% editing two days post-electroporation (FIG. 8A).Editing was also detected in human CD34+ cells isolated at 9 and 20weeks post-transplantation (FIGS. 27B and 27C).

Gene-Editing, Long-Term Engrafted HSCs Sustained Increased Production ofFetal Hemoglobin

NSG mice do not support erythroid development since the mouse lack theright human cytokine to enable erythroid maturation. However, engraftedhuman HSCs can be harvested from the mouse bone marrow, placed inerythroid differentiation medium (as described elsewhere in theseexamples) to induce erythroid differentiation. Data show thatgene-edited, 20 weeks engrafted HSCs were capable of producing higherlevel of HbF compare to mock edited and transplanted HSCs (FIG. 28).

Example 7. Off Target Indel Pattern Analysis

In Silico Identification of Potential gRNA Off-Target Loci

Potential off-target loci for the subset of HGB1/HBG2 region gRNAsGCR-0001, GCR-0008, GCR-0010, GCR-0048, GCR-0051 and GCR-0067 wereidentified as follows. For each gRNA, the 20 nucleotide gRNA targetingdomain sequence was aligned to the human genome reference sequence(build GRCh38) using the BFAST sequence aligner (version 0.6.4f, Homeret al, PLoS One, 2009, 4(11), e7767, PMID: 19907642) using standardparameters allowing up to 5 nucleotide mismatches. Loci identified werefiltered to only contain sites that are 5′ adjacent to the Cas9canonical 5′-NGG-3′ PAM sequence (i.e. 5′-off-target locus-PAM-3′).Using the BEDTools script (version 2.11.2, Quinlan and Hall,Bioinformatics, 2010 26(6):841-2, PMID: 20110278) sites with 5nucleotide mismatches were further filtered against RefSeq geneannotations (Pruitt et al, Nucleic Acids Res., 2014 42 (Databaseissue):D756-63, PMID: 24259432) to only contain loci annotated as exons.Counts of the potential off-target loci identified for the HGB1/HBG2region gRNAs are shown in Table 12.

TABLE 12 Counts of in silico off-target loci identified for theHBG1/HBG2 region gRNAs GCR-0001, GCR-0008, GCR-0010, GCR-0048, GCR-0051and GCR-0067 with 0, 1, 2, 3 and 4 nucleotide mismatches and 5nucleotide mismatches within RefSeq exons are shown. Number ofoff-targets with N mismatches gRNA name 0 1 2 3 4 5 RefSeq exons Totalsites GCR-0001 0 0 4⁺ 17 323 28 372 GCR-0008 0 1⁺ 0 27 267 60 355GCR-0010 0 1⁺ 0 17 216 64 298 GCR-0048* 0 0 0 7 89 38 134 GCR-0051 0 1⁺3 25 369 66 464 GCR-0067* 0 0 0 11 95 38 144 *gRNAs GCR-0048 andGCR-0067 have two perfect match on-target sites one per HBG locus.⁺includes one or two mismatch homologous HBG1 or HBG2 target sites.

Off-Target Analysis in CD34+ HSPCs

Genomic DNA Extraction

Genomic DNA was isolated from 3 day RNP edited and unedited bone marrowderived CD34+ HSPC using the Quick-DNA Miniprep kit (Zymo Research)following the manufacturer's recommendations.

PCR Primer Design for Targeted Amplification of Potential Off-TargetSites

PCR amplicons targeting potential off-target loci with 0-3 mismatches(and the on-target locus) identified for the HBG1/HBG2 region gRNAs(GCR-0001, GCR-0008, GCR-0010, GCR-0048, GCR-0051 and GCR-0067) weredesign using Primer3 (version 2.3.6, Untergasser et al, Nucleic AcidsRes., 2012 40(15):e115, PMID: 22730293) using default parameters aimingfor an amplicon size range of approximately 160-300 base pairs in lengthwith the gRNA targeting domain sequence located in the center of theamplicon. Resulting PCR primer pairs and amplicon sequences were checkedfor uniqueness by BLAST searching (version 2.2.19, Altschul et al, J MolBiol., 1990, 215(3):403-10, PMID: 2231712) sequences against the humangenome reference sequence (build GRCh38). Primer pairs resulting in morethan one amplicon sequence were discarded and redesigned. Table 3 showscounts of successful PCR primer pairs designed.

Illumina Sequencing Library Preparation, Quantification and Sequencing

Genomic DNA from RNP edited (2 replicates per gRNA) and unedited (2replicate per gRNA) HSPC was quantified using the Quant-iT PicoGreendsDNA kit (Thermo Fisher, Cat #P7581) using manufacture'srecommendations. Illumina sequencing libraries targeting individualoff-target loci (and the on-target locus) were generated for each sampleusing two sequential PCR reactions. The first PCR amplified the targetlocus using target specific PCR primers (designed above) that weretailed with universal Illumina sequencing compatible sequences. Thesecond PCR added additional Illumina sequencing compatible sequences tothe first PCR amplicon, including sample barcodes to enable multiplexingduring sequencing. PCR 1 was performed in a final volume of 10 μL witheach reaction containing approximately 6.5 ng of gDNA (equivalent toapproximately 1000 cells), PCR 1 primer pairs (Integrated DNATechnologies) at a final concentration of 0.25 μM and 1× finalconcentration of Q5 Hot Start Master Mix (New England BioLabs, Cat#102500-140). PCR 1 left primers were 5′ tailed (i.e. 5′-tail-targetspecific left primer-3′) with sequence5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′ (SEQ ID NO: 279) and rightprimers were 5′ tailed (i.e. 5′-tail-target specific right primer-3′)with sequence 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′ (SEQ ID NO: 280).PCR 1 was performed on a thermocycler using the following cyclingconditions: 1 cycle of 98° C. for 1 min; 25 cycles of 98° C. for 10 sec,63° C. for 20 sec, and 72° C. for 30 sec; 1 cycle at 72° C. for 2 min.PCR 1 was then diluted 1 in 100 using nuclease free water (Ambion, Cat#AM9932) and used as input into PCR 2. PCR 2 was performed in a finalvolume of 10 μL with each reaction containing 2 μL of diluted PCR 1product, PCR 2 primer pairs (Integrated DNA Technologies) at a finalconcentration of 0.5 μM and 1× final concentration of Q5 Hot StartMaster Mix (New England BioLabs, Cat #102500-140). PCR 2 left primersequence used was5′-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTC-3′ (SEQ ID NO:281) and PCR 2 right primer sequence used was5′-CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGG-3′ (SEQ ID NO: 282)where the NNNNNNNN denote an 8 nucleotide barcode sequence used forsample multiplexing as part of the standard Illumina sequencing process.PCR 2 was performed on a thermocycler using the following cyclingconditions: 1 cycle of 72° C. for 3 min; 1 cycle of 98° C. for 2 min; 15cycles of 98° C. for 10 sec, 63° C. for 30 sec, and 72° C. for 2 min.PCR 2 amplicons, now viable Illumina sequencing libraries, were cleanedup using Agencourt AMPure XP beads (Beckman Coulter, Cat #A63882)following the manufacture's recommendations. The cleaned Illuminasequencing libraries were then quantified using standard qPCRquantification methods using Power SYBR Green PCR master mix (LifeTechnologies, Cat #4367660) and primers specific to the Illuminasequencing library ends (forward primer sequence5′-CAAGCAGAAGACGGCATACGA-3′ (SEQ ID NO: 283) and reverse primer sequence5′-AATGATACGGCGACCACCGAGA-3′ (SEQ ID NO: 284)). Illumina sequencinglibraries were then pooled equimolar and subjected to Illuminasequencing on a MiSeq instrument (Illumina, Cat #SY-410-1003) with 300base paired-end reads using a MiSeq Reagent Kit v3 (Illumina, Cat#MS-102-3003) following the manufacture's recommendations. A minimum of1000-fold sequence coverage was generated for each locus in eachreplicate. PCR, cleanup, pooling and sequencing of edited and uneditedsamples were performed separately to avoid any possibility of crosscontamination between samples or PCR amplicons generated therefrom.

NGS Sequencing Data QC and Variant Analysis

Methods are as in Example 2.1 with the following modifications. ForStage 5 of analysis, sites with a combined indel frequency of >2%(editing in more than approximately 10-20 cell) were considered andpotential active editing sites were further examined at the readalignment level using the Integrative Genome Viewer (IGV version 2.3,Robinson et al, Nat Biotechnol. 2011, 9(1):24-6, PMID: 21221095) thatallows for visual inspection of read alignments to the genome referencesequence.

Quantification of Large Deletions.

Large deletions between targeting sites at the HBG1 and HBG2 promotorswere quantified by digital droplet PCR (ddPCR) in non-competitive assayformat for copy number determination according to manufacturer'srecommendations. Briefly, gDNA was combined with ddPCR SuperMix forProbes (no dUTPs) (BioRad Cat #1863024), HindIII-HF restriction enzyme(NEB Cat #R3104S) and each primer probe mix, transferred to a DG8cartridge along with Droplet Generation Oil for Probes (BioRad Cat#1863005) and droplets generated with a QX200 droplet generator(BioRad). Droplets were subject to PCR on a C1000 Touch Thermal Cyclerwith 96-Deep Well Reaction Module (BioRad), followed by detection withQX200 droplet reader (BioRad). Copies per ul was determined by analysiswith QuantaSoft software (BioRad). A custom primer probe set (LifeTechnologies Cat #APZW76R, PN4331348, Forward primer:ACGGATAAGTAGATATTGAGGTAAGC (SEQ ID NO: 285), Reverse primer:GTCTCTTTCAGTTAGCAGTGG (SEQ ID NO: 286), FAM TaqMan Probe:ACTGCGCTGAAACTGTGGCTTTATAG (SEQ ID NO: 287)) used to amplify gDNA withinthe HBG1-HBG2 intergenic region. A TaqMan Copy Number Reference Assay,human, RNase P (Thermo Fisher cat #4403326) was used as a referenceamplicon. Copies per ul for HBG1-HBG2 and RNase P amplicons were withinthe manufacturer's reported linear range. Percent deletion was reportedas 100% times 1 minus the ratio of copies per ul for HBG1-HBG2 and RNaseP amplicons. Unedited control samples had calculated percent deletionsup to 2%, which may reflect background in the assay.

HBG1/HBG2 Region in Silico Off-Target Analysis Results

Table 13 shows the number of off-target sites successfullycharacterized. Uncharacterized sites failed in PCR primer design or PCRamplification and remain to be evaluated.

gRNA GCR-0001: The HBG1 on-target site showed robust localized editingwith an average INDEL frequency of approximately 58%, whereas thehomologous HBG2 target site with two mismatches (here and below, shownin lowercase letters) relative to gRNA targeting domain sequence(5′-AGTCCTGGTATCtTCTATGg-PAM-3′, PAM=TGG (SEQ ID NO: 288)) showedminimal localized editing. However, ddPCR analysis of the 4.9 kbdeletion showed it occurring at a frequency of approximately 34%.Further analysis identified one positive off-target site with an averageINDEL frequency of approximately 26% in both replicates. The site has 3mismatches relative to the gRNA targeting domain sequence(5′-AtTCCcaGTATCCTCTATGA-PAM-3′, PAM=TGG (SEQ ID NO: 289)) and islocated in an intergenic on the Y chromosome at base pair position21,470,475-21,470,497. It is unclear whether editing at this off-targetsite has any detrimental effect on gene expression or cell viability,further analysis is required.

gRNA GCR-0008: The HBG1 on-target site showed robust localized editingwith an average INDEL frequency of approximately 88%. The homologousHBG2 target site with one mismatch relative to the gRNA targeting domainsequence (5′-GGAGAAGaAAACTAGCTAAA-PAM-3′, PAM=GGG (SEQ ID NO: 290))showed robust localized editing with an average INDEL frequency ofapproximately 85%. ddPCR analysis of the 4.9 kb deletion showed itoccurring at a frequency of approximately 50%. No other sites showedediting.

gRNA GCR-0010: The HBG1 on-target site showed robust localized editingwith an average INDEL frequency of approximately 32%. The homologousHBG2 target site with one mismatch relative to the gRNA targeting domainsequence (5′-GGGAGAAGaAAACTAGCTAA-PAM-3′, PAM=AGG (SEQ ID NO: 291))showed robust localized editing with an average INDEL frequency ofapproximately 27%. ddPCR analysis of the 4.9 kb deletion showed itoccurring at a frequency of approximately 33%. No other sites showedediting.

gRNA GCR-0048: The HBG1 and HBG2 on-target sites showed robust localizedediting with an average INDEL frequency of approximately 86% for bothsites. ddPCR analysis of the 4.9 kb deletion showed it occurring at afrequency of approximately 45%. No other sites showed editing.

gRNA GCR-0051: The HBG2 on-target site showed robust localized editingwith an average INDEL frequency of approximately 88%. The homologousHBG1 target site with one mismatch relative to the gRNA targeting domainsequence (5′-GGAGAAGgAAACTAGCTAAA-PAM-3′, PAM=GGG (SEQ ID NO: 292))showed robust localized editing with an average INDEL frequency ofapproximately 59%. ddPCR analysis of the 4.9 kb deletion showed itoccurring at a frequency of approximately 40%. No other sites showedediting.

gRNA GCR-0067: The HBG1 and HBG2 on-target sites showed robust localizedediting with an average INDEL frequency of approximately 74% and 78%respectively. ddPCR analysis of the 4.9 kb deletion showed it occurringat a frequency of approximately 62%. No other sites showed editing.

The localized INDEL frequencies described above are not relative to thetotal indel frequency and do not take into account the frequency of thelarge 4.9 kb deletion.

TABLE 13 Counts of in silico 0-3 mismatch off-target sites identifiedfor the HBG1/HBG2 region gRNAs GCR-0001, GCR-0008, GCR- 0010, GCR-0048,GCR-0051 and GCR-0067, counts of sites successfully characterized ingenome-edited HSPC and counts of sites that show editing are shown.Number of in Number of active Number of 0-3 silico sites in silicomismatch in silico successfully off-target sites gRNA name off-targetsites characterized identified GCR-0001 21 19 1 GCR-0008 28 28 1 (HBG2)GCR-0010 18 18 1 (HBG2) GCR-0048 7 7 0 GCR-0051 29 29 1 (HBG1) GCR-006711 11 0

Unbiased Off-Target Analysis

An oligo insertion based assay (See, e.g., Tsai et al., NatureBiotechnology. 33, 187-197; 2015) was used to determine potentialoff-target genomic sites cleaved by Cas9 targeting HBG1 and/or HBG2. Inthese experiments, Cas9GFP-expressing HEK293 cells (HEK-293_Cas9GFP)were transfected with gRNAs (15 nM crRNA:tracr) and insertion oligo (10nM) using Lipofectamine® RNAiMAX. The assay relies on the identificationof the oligo incorporated into double stranded breaks in the genome,which may or may not result from cleavage by Cas9.

In one experiment, gRNAs (dual guide RNAs comprising the indicatedtargeting domain in FIG. 29) targeting HBG1 and/or HBG2 were screened inthe HEK-293_Cas9GFP cells, and the results are plotted in FIG. 29. In aseparate experiment, the same methodology was used to screen some of thesame as well as additional gRNAs (including dual guide and single guideRNAs comprising the indicated targeting domain in FIG. 30) targetingHBG1 and/or HBG2 in the HEK-293_Cas9GFP cells, and the results areplotted in FIG. 30. The experiment with data depicted in FIG. 30 used analternative insertion oligo with a balanced G/C content and minimizedcomplementarity with the human genome, as well as other modifications tothe PCR steps as compared to the published Tsai et al. methods, whichimproved sensitivity and diminished false positives. In bothexperiments, the assay detected high-efficiency editing at the expectedtarget sequences and one or more potential off-target sites.

While the detection of the insertion oligo at sites in the genome otherthan the on-target site identifies potential off-target effects of Cas9,targeted deep sequencing of the potential off-target sites may be usedto determine whether the potential sites are bona fide off-target sitescleaved by Cas9. To this end, an experiment was performed in which theHEK-293_Cas9GFP cells were similarly transfected with the gRNAs (at 25nM crRNA:tracr) used in the previous two experiments, but without theinsertion oligo. Amplicon deep sequencing of each of the identifiedpotential off-target sites depicted in FIGS. 29 and 30 was used toidentify whether indels (indicative of Cas9 cleavage events) werepresent at the potential off-target sites in the HEK-293_Cas9GFP cellline. The results of this experiment demonstrated that most of thepotential off-target sites identified by the oligo insertion assay didnot have detectable indels following transfection of the gRNAs, with afew exceptions as provided in Table 14 below. Of note, theHEK-293_Cas9GFP cells used in these experiments for detecting potentialoff-targets constitutively overexpress Cas9, likely leading to a highernumber of potential off-target “hits” as compared to a transientdelivery modality (e.g., RNP delivery) in various cell types of interest(e.g., CD34+ HSCs).

TABLE 14 Off-target sites validated in the HEK-293_Cas9GFP cell line.Average Indel Guide ID Off-target Coordinate Frequency CR005813 chr13:95591406-95591426    2% (dgRNA; GCR-0051) G000690 chr13:95591406-95591426    2% (sgRNA GCR-0051) CR005821 chr20:10409602-10409622 27.60% (dgRNA GCR008) G000692 chr20: 10409602-1040962220.10% (sgRNA GCR008)

Using the methods described herein, potential off-target sites wereexamined in sgRNA/Cas9 edited CD34+ HSPCs and showed no editing in theCD34+ cell type.

Example 8

Experimental Procedure

CD34+ cells harvested from the mobilized peripheral blood (mPB) ofhealthy donors were cultured and gene edited in the same condition asdescribed previously in Example 6. Cells were characterized for theirbiological function using the same methods described in previousparagraphs.

CD34+ Cells Derived from Mobilized Peripheral Blood Maintain their CD34+Cell Count, Expansion Capacity, and Viability Upon Editing

The number of CD34+ cells transplanted into patient is directlycorrelated with the success of a bone marrow transplant. Therefore, weenumerated the percentage of CD34+ cells over a 10-day period upongene-editing using a clinically acceptable method, ISHAGE. Results showthat neither editing with sg1128 nor sg0067 impacted CD34+ cell count(FIG. 31) when compared to mock edited control. For the total durationof our cell process, in which cells were only maintained in culture for3 days after electroporation, the percentage of CD34+ cells wasmaintained at approximately 90%. (FIG. 31A). Gene editing also did notimpact the capacity of CD34+ cells to expand (FIG. 31B) and theirviability post-electroporation ranged between 70-90% (FIG. 31C). Weobserved a ˜2 fold cell expansion at day 3 post-electroporation, ˜10fold expansion at day 7 post-electroporation, and 15-25 fold expansionat day 10 after electroporation (FIG. 31B).

CD34+ Cells Derived from Mobilized Peripheral Blood of HealthyIndividuals Demonstrate Similar Editing Efficiencies Compared to BoneMarrow Cell Source or CD34+ Cells from Sickle Cell Disease Patients

Sg1128 demonstrated about 70-75% editing efficiency in CD34+ cellsderived from mPB of healthy donors, whereas sg0067 showed >95% totalediting efficiency (including large 5 kb deletion and small indels)(FIG. 32). Editing efficiencies from these sgRNA were similar in cellsfrom different sources, including bone marrow derived CD34+ cells fromhealthy donors or peripheral blood derived CD34+ cells from sickle celldisease patients (see other examples). It is important to note that boththe editing efficiency and the editing pattern of each sgRNA were highlyconsistent across all donors tested.

CRISPR knockdown of BCL11A or mutation of the g-globin gene clusterincreases g-globin transcript and F-cell production

Knockdown of BCL11A by sg1128, or mutating the potential BCL11A bindingsite at the g-globin gene cluster by sg0067, both significantlyaugmented g-globin transcripts (FIG. 33) leading to 15-20% upregulationof F-cell production compared to mock edited control (FIG. 34). Insummary, sg1128 and sg0067 can edit CD34+ cells with high efficiency andgenerate highly consistent editing patterns. Edited cells maintainapproximately 90% CD34+ cell count by the end of our 6 days cellprocessing procedure. The expansion capacity and viability of cells werenot impaired by the gene editing procedure. Edited CD34+ cells, whendifferentiated into erythrocytes, expressed significantly higher levelof g-globin transcripts, translating to an increased number of F-cellproduction. This improved hemoglobin expression and F-cell number mayrescue the hematological features of sickle cell disease.

Example 9: In Vivo Engraftment and Characterization of Gene Edited HspcsDerived from Sickle Cell Disease Patients

Experimental Procedure

CD34+ cells from sickle cell disease patients were cultured and geneedited in the same condition as described previously in Example 8. Cellswere characterized for their biological function using the same methodsdescribed in previous Examples.

CD34+ Cells Derived from Sickle Cell Disease Individuals Maintain theirCD34+ Immunophenotype and Viability Upon Editing.

Hematopoietic stem and progenitor cells (HSPCs) maintaining theirprimitive cell state should express CD34. This is one of the mostimportant clinical markers that associates with engraftablehematopoietic stem cell upon transplantation, and the success of atransplant is directly correlated with the number of CD34+ cellstransplanted. Therefore, we enumerated the number of CD34+ cellsobtained from the peripheral blood of sickle individuals upon editingusing a clinically acceptable method, ISHAGE. Results show that neitherediting with sg1128 nor sg0067 impacted CD34+ cell count (FIG. 35A andFIG. 35B) when compared to mock edited control. The percentage of CD34+cells were maintained at ˜80% at day 3 upon editing (FIG. 35B). Geneediting also did not impact the capacity of patient derived CD34+ cellsto expand (FIG. 35C) and their viability post-electroporation(electroporation at D0) was over 75% at all time points measured over a10-day period (FIG. 35D).

CD34+ Cells Derived from Sickle Cell Disease Individuals DemonstrateSimilar Editing Efficiencies Compared to CD34+ Cells from Healthy Donors

Sg1128 demonstrated about 65% editing efficiency in CD34+ cells derivedfrom sickle cell patients, whereas sg0067 showed 80-95% editingefficiency in patient samples (FIG. 36). The level of editing efficiencyis similar to those obtained using either bone marrow or mobilizedperipheral blood derived CD34+ cells from healthy donors. Editingpattern of each guide RNA as measured by NGS (as described in Example2.1) were also highly consistent across different patients.

Gene Editing does not Compromise the In Vitro Multi-LineageDifferentiation Capacity of CD34+ Cells Derived from Sickle Cell DiseaseIndividuals

Gene edited hematopoietic stem and progenitor cells were fully capableof differentiating into erythroid, granulocytic, monocytic, andmegakaryocytic lineages as measured by colony-forming unit assays (FIG.37).

CRISPR Knockdown of BCL11A or Mutation of the g-Globin Gene ClusterIncreases g-Globin Transcript, F-Cell Production, and Fetal HemoglobinExpression

Either knockdown of BCL11A by sg1128, or creating indels/deletions atthe g-globin cluster by sg0067, significantly augmented g-globintranscripts (FIG. 38) and increased the number of F-cells as measured byflow cytometry (FIG. 39). In addition, the fetal hemoglobin expressionintensity of the F-cells was also improved on a per cell basis asmeasured by flow cytometry (FIG. 40).

Gene Editing of Patient Derived CD34+ Cells LED to Significant Decreasein Sickle Cell Count and Increase in Normal Cell Number

Finally, to understand whether gene editing can rescue the sickle cellmorphology, we CRISPR-edited sickle cell disease patient derived CD34+cells, differentiated these cells into red blood cells, and subjectedthese cells into a hypoxia chamber for 4 days to induce the sickle cellmorphology. Cells were co-stained with anti-HbF-FITC antibody, fixedwithin the chamber, and subjected to imaging flow cytometry to captureone single image per cell. Single cell imaging flow cytometry candistinguish sickle cell versus normal cell based on cell length andexpression of HbF in a high throughput manner (40,000 single cell imagesfrom each patient were used for data analysis). Results show that geneediting with either sg1128 or sg0067 were able to decrease sickle cellcount by approximately 40% (FIG. 41A) and concomitantly increase normalcell count by 1.2 fold (FIG. 41B). The compound effect of decreasingsickle cell count and concurrently increasing normal red blood cellcount will greatly benefit patients when translated to the clinic.

In summary, sg1128 and sg0067 can edit CD34+ cells from sickle celldisease patients with high efficiency and generate highly consistentediting patterns. The percentage of CD34+ cells, cell expansioncapacity, and viability of cells were not impaired by the gene editingprocedure when comparing edited cells to mock edited control group.Edited CD34+ cells, when differentiated into erythrocytes, expressedsignificantly higher level of g-globin transcripts. This translates toan increased number of F-cell and also augmented HbF expression percell. The increase in the number of high HbF-expressing F-cell andreduction in the number of sickle red blood cell was reflected in oursingle cell imaging flow cytometry analysis. We observed a 50% decreaseof sickle cells in gene edited group and simultaneously a 1.2 foldincrease of high HbF-expressing normal red blood cells in edited groupswhen compared to mock edited control. This compound effect of reducingsickle cell number with a concomitant increase in high HbF-expressingnormal red blood cell upon gene editing should significantly benefitpatients when translated to the clinic. Together, these data support thedevelopment of CRISPR/Cas-mediated genome editing as a means of celltherapy to treat b-globinopathies.

To the extent there are any discrepancies between any sequence listingand any sequence recited in the specification, the sequence recited inthe specification should be considered the correct sequence. Unlessotherwise indicated, all genomic locations are according to hg38.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. While this invention hasbeen disclosed with reference to specific aspects, it is apparent thatother aspects and variations of this invention may be devised by othersskilled in the art without departing from the true spirit and scope ofthe invention. The appended claims are intended to be construed toinclude all such aspects and equivalent variations.

The invention claimed is:
 1. A guide RNA (gRNA) molecule comprising,from 5′ to 3′, [targeting domain]-SEQ ID NO: 195, wherein the targetingdomain comprises SEQ ID NO:
 67. 2. The gRNA molecule of claim 1, whereinsaid SEQ ID NO: 195 is disposed immediately 3′ to said targeting domain.3. The gRNA molecule of claim 1, further comprising, at the 3′ end, 1,2, 3, 4, 5, 6 or 7 uracil (U) nucleotides.
 4. A guide RNA (gRNA)molecule comprising, from 5′ to 3′, [targeting domain]-SEQ ID NO: 231,wherein the targeting domain comprises SEQ ID NO:
 67. 5. The gRNAmolecule of claim 4, wherein said SEQ ID NO: 231 is disposed immediately3′ to said targeting domain.
 6. The gRNA molecule of claim 1, comprisingthe sequence of: (a) SEQ ID NO: 174; (b) SEQ ID NO: 175; or (c) SEQ IDNO:
 176. 7. The gRNA molecule of claim 1, wherein a) when a CRISPRsystem comprising the gRNA molecule is introduced into a cell, an indelis formed at or near a target sequence complementary to the targetingdomain of the gRNA molecule; and/or b) when a CRISPR system comprisingthe gRNA molecule is introduced into a cell, a deletion is createdcomprising a sequence between a sequence complementary to the gRNAtargeting domain in the HBG1 promoter region and a sequencecomplementary to the gRNA targeting domain in the HBG2 promoter region.8. A composition comprising: 1) the gRNA molecule of claim 1 and a Cas9molecule; 2) the gRNA molecule of claim 1 and a polynucleotidecomprising a nucleic acid sequence encoding a Cas9 molecule; 3) apolynucleotide comprising a nucleic acid sequence encoding the gRNAmolecule of claim 1 and a Cas9 molecule; 4) a polynucleotide comprisinga nucleic acid sequence encoding the gRNA molecule of claim 1 and apolynucleotide comprising a nucleic acid sequence encoding a Cas9molecule; 5) a polynucleotide comprising a nucleic acid sequenceencoding the gRNA molecule of claim 1; 6) any of 1) to 5), above, and atemplate nucleic acid; or 7) any of 1) to 5) above, and a polynucleotidecomprising a nucleic acid sequence encoding a template nucleic acid. 9.A composition comprising the gRNA molecule of claim 1, furthercomprising a Cas9 molecule, wherein the Cas9 molecule comprises thesequence of SEQ ID NO: 205 or a sequence with at least 95% sequencehomology thereto.
 10. A nucleic acid sequence that encodes the gRNAmolecule of claim
 1. 11. A vector comprising the nucleic acid sequenceof claim 10, wherein said vector is selected from the group consistingof a lentiviral vector, an adenoviral vector, an adeno-associated viral(AAV) vector, a herpes simplex virus (HSV) vector, a plasmid, aminicircle, a nanoplasmid, and an RNA vector.
 12. A cell, comprising thegRNA molecule of claim 1 and a Cas9 molecule.
 13. The gRNA molecule ofclaim 1, consisting of the sequence of: (a) SEQ ID NO: 174; (b) SEQ IDNO: 175; or (c) SEQ ID NO:
 176. 14. The gRNA molecule of claim 1,consisting of the sequence of SEQ ID NO:
 174. 15. The composition ofclaim 9, wherein the Cas9 molecule comprises the sequence of: (a) SEQ IDNO: 233; (b) SEQ ID NO: 234; (c) SEQ ID NO: 235; (d) SEQ ID NO: 236; (e)SEQ ID NO: 237; (f) SEQ ID NO: 238; (g) SEQ ID NO: 239; (h) SEQ ID NO:240; (i) SEQ ID NO: 241; (j) SEQ ID NO: 242; (k) SEQ ID NO: 243; or (l)SEQ ID NO:
 244. 16. The composition of claim 9, wherein the Cas9molecule comprises the sequence of SEQ ID NO:
 244. 17. The compositionof claim 9, formulated in a medium suitable for electroporation.